Pro104 antibody compositions and methods of use

ABSTRACT

The invention provides isolated anti-ovarian, pancreatic, lung or breast cancer antigen (Pro104) antibodies that bind to Pro 104 on a mammalian cell in vivo. The invention also encompasses compositions comprising an anti-Pro104 antibody and a carrier. These compositions can be provided in an article of manufacture or a kit Another aspect of the invention is an isolated nucleic acid encoding an anti-Pro 104 antibody, as well as an expression vector comprising the isolated nucleic acid. Also provided are cells that produce the anti-Pro 104 antibodies. The invention encompasses a method of producing the anti-Pro 104 antibodies. Other aspects of the invention are a method of killing a Pro 104-expressing cancer cell, comprising contacting the cancer cell with an antiPro104 antibody and a method of alleviating or treating a Pro 104-expressing cancer in a mammal, comprising administering a therapeutically effective amount of the anti-Pr 104 antibody to the mammal.

This patent application claims the benefit of priority from U.S.Provisional Patent Application No. 60/523,271, filed Nov. 17, 2003 andU.S. Provisional Patent Application No. 60/485,346, filed Jun. 27, 2003,each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-Pro104 antibody compositions andmethods of detecting Pro104 expressing cancers and killingPro104-expressing breast, ovarian pancreatic and lung cancers cells. Inaddition, this invention relates to methods of modulating or killing aPro104 expressing cell by administering an effective amount of acompound capable of modulating Pro104 function.

BACKGROUND OF THE INVENTION

Breast Cancer

Breast cancer, also referred to as mammary tumor cancer, is the secondmost common cancer among women, accounting for a third of the cancersdiagnosed in the United States. One in nine women will develop breastcancer in her lifetime and about 192,000 new cases of breast cancer arediagnosed annually with about 42,000 deaths. Bevers, Primary Preventionof Breast Cancer, in Breast Cancer, 20-54 (Kelly K Hunt et al., ed.,2001); Kochanek et al., 49 Nat'l. Vital Statistics Reports 1, 14 (2001).Breast cancer is extremely rare in women younger than 20 and is veryrare in women under 30. The incidence of breast cancer rises with ageand becomes significant by age 50. White Non-Hispanic women have thehighest incidence rate for breast cancer and Korean women have thelowest. Increased prevalence of the genetic mutations BRCA1 and BRCA2that promote breast and other cancers are found in Ashkenazi Jews.African-American women have the highest mortality rate for breast canceramong these same groups (31 per 100,000), while Chinese women have thelowest at 11 per 100,000. Although men can get breast cancer, this isextremely rare. In the United States it is estimated there will be217,440 new cases of breast cancer and 40,580 deaths due to breastcancer in 2004. (American Cancer Society Website: cancer with theextension.org of the world wide web). With the exception of those caseswith associated genetic factors, precise causes of breast cancer are notknown.

In the treatment of breast cancer, there is considerable emphasis ondetection and risk assessment because early and accurate staging ofbreast cancer has a significant impact on survival. For example, breastcancer detected at an early stage (stage T0, discussed below) has afive-year survival rate of 92%. Conversely, if the cancer is notdetected until a late stage (i.e., stage T4 (IV)), the five-yearsurvival rate is reduced to 13%. AJCC Cancer Staging Handbook pp. 164-65(Irvin D. Fleming et al. eds., 5th ed. 1998). Some detection techniques,such as mammography and biopsy, involve increased discomfort, expense,and/or radiation, and are only prescribed only to patients with anincreased risk of breast cancer.

Current methods for predicting or detecting breast cancer risk are notoptimal. One method for predicting the relative risk of breast cancer isby examining a patient's risk factors and pursuing aggressive diagnosticand treatment regiments for high risk patients. A patient's risk ofbreast cancer has been positively associated with increasing age,nulliparity, family history of breast cancer, personal history of breastcancer, early menarche, late menopause, late age of first full termpregnancy, prior proliferative breast disease, irradiation of the breastat an early age and a personal history of malignancy. Lifestyle factorssuch as fat consumption, alcohol consumption, education, andsocioeconomic status have also been associated with an increasedincidence of breast cancer although a direct cause and effectrelationship has not been established. While these risk factors arestatistically significant, their weak association with breast cancerlimited their usefulness. Most women who develop breast cancer have noneof the risk factors listed above, other than the risk that comes withgrowing older. NIH Publication No. 00-1556 (2000).

Current screening methods for detecting cancer, such as breastself-exam, ultrasound, and mammography have drawbacks that reduce theireffectiveness or prevent their widespread adoption. Breast self-exams,while useful, are unreliable for the detection of breast cancer in theinitial stages where the tumor is small and difficult to detect bypalpation. Ultrasound measurements require skilled operators at anincreased expense. Mammography, while sensitive, is subject to overdiagnosis in the detection of lesions that have questionable malignantpotential. There is also the fear of the radiation used in mammographybecause prior chest radiation is a factor associated with an increaseincidence of breast cancer.

At this time, there are no adequate methods of breast cancer prevention.The current methods of breast cancer prevention involve prophylacticmastectomy (mastectomy performed before cancer diagnosis) andchemoprevention (chemotherapy before cancer diagnosis) which are drasticmeasures that limit their adoption even among women with increased riskof breast cancer. Bevers, supra.

A number of genetic markers have been associated with breast cancer.Examples of these markers include carcinoembryonic antigen (CEA) (Mughalet al., JAMA 249:1881 (1983)), MuC-1 (Frische and Liu, J. Clin. Ligand22:320 (2000)), HER-2/neu (Haris et al., Proc. Am. Soc. Clin. Oncology15:A96 (1996)), uPA, PAI-1, LPA, LPC, RAK and BRCA (Esteva and Fritsche,Serum and Tissue Markers for Breast Cancer, in Breast Cancer 286-308(2001)). These markers have problems with limited sensitivity, lowcorrelation, and false negatives which limit their use for initialdiagnosis. For example, while the BRCA1 gene mutation is useful as anindicator of an increased risk for breast cancer, it has limited use incancer diagnosis because only 6.2% of breast cancers are BRCA1 positive.Malone et al, JAMA 279:922 (1998). See also, Mewman et al., JAMA 279:915(1998) (correlation of only 3.3%).

There are four primary classifications of breast cancer varying by thesite of origin and the extent of disease development.

-   -   I. Ductal carcinoma in situ (DCIS): Malignant transformation of        ductal epithelial cells that remain in their normal position.        DCIS is a purely localized disease, incapable of metastasis.    -   II. Invasive ductal carcinoma (DC): Malignancy of the ductal        epithelial cells breaking through the basal membrane and into        the supporting tissue of the breast. IDC may eventually spread        elsewhere in the body.    -   III. Lobular carcinoma in situ (LCIS): Malignancy arising in a        single lobule of the breast that fails to extend through the        lobule wall, it generally remains localized.    -   IV. Infiltrating lobular carcinoma (ILC): Malignancy arising in        a single lobule of the breast and invading directly through the        lobule wall into adjacent tissues.

By virtue of its invasion beyond the lobule wall, ILC may penetratelymphatics and blood vessels and spread to distant sites.

For purpose of determining prognosis and treatment, these four breastcancer types have been staged according to the size of the primary tumor(I), the involvement of lymph nodes (N), and the presence of metastasis(M). Although DCIS by definition represents localized stage I disease,the other forms of breast cancer may range from stage II to stage IV.There are additional prognostic factors that further serve to guidesurgical and medical intervention. The most common ones are total numberof lymph nodes involved, ER (estrogen receptor) status, Her2/neureceptor status and histologic grades.

Breast cancers are diagnosed into the appropriate stage categoriesrecognizing that different treatments are more effective for differentstages of cancer. Stage TX indicates that primary tumor cannot beassessed (i.e., tumor was removed or breast tissue was removed). StageT0 is characterized by abnormalities such as hyperplasia but with noevidence of primary tumor. Stage Tis is characterized by carcinoma insitu, intraductal carcinoma, lobular carcinoma in situ, or Paget'sdisease of the nipple with no tumor. Stage T1 (I) is characterized ashaving a tumor of 2 cm or less in the greatest dimension. Within stageT1, Tmic indicates microinvasion of 0.1 cm or less, T1a indicates atumor of between 0.1 to 0.5 cm, T1b indicates a tumor of between 0.5 to1 cm, and T1c indicates tumors of between 1 cm to 2 cm. Stage T2 (II) ischaracterized by tumors from 2 cm to 5 cm in the greatest dimension.Tumors greater than 5 cm in size are classified as stage T3 (E). StageT4 (IV) indicates a tumor of any size with extension to the chest wallor skin. Within stage T4, T4a indicates extension of the tumor to thechess wall, T4b indicates edema or ulceration of the skin of the breastor satellite skin nodules confined to the same breast, T4c indicates acombination of T4a and T4b, and T4d indicates inflammatory carcinoma.AJCC Cancer Staging Handbook pp. 159-70 (Irvin D. Fleming et al. eds.,5^(th) ed. 1998). In addition to standard staging, breast tumors may beclassified according to their estrogen receptor and progesteronereceptor protein status. Fisher et al., Breast Cancer Research andTreatment 7:147 (1986). Additional pathological status, such as HER2/neustatus may also be useful. Thor et al., J. Nat'l. Cancer Inst. 90:1346(1998); Paik et al., J. Nat'l. Cancer Inst. 90:1361 (1998); Hutchins etal., Proc. Am. Soc. Clin. Oncology 17:A2 (1998); and Simpson et al., J.Clin. Oncology 18:2059 (2000).

In addition to the staging of the primary tumor, breast cancermetastases to regional lymph nodes may be staged. Stage NX indicatesthat the lymph nodes cannot be assessed (e.g., previously removed).Stage N0 indicates no regional lymph node metastasis. Stage N1 indicatesmetastasis to movable ipsilateral axillary lymph nodes. Stage N2indicates metastasis to ipsilateral axillary lymph nodes fixed to oneanother or to other structures. Stage N3 indicates metastasis toipsilateral internal mammary lymph nodes. Id.

Stage determination has potential prognostic value and provides criteriafor designing optimal therapy. Simpson et al., J. Clin. Oncology 18:2059(2000). Generally, pathological staging of breast cancer is preferableto clinical staging because the former gives a more accurate prognosis.However, clinical staging would be preferred if it were as accurate aspathological staging because it does not depend on an invasive procedureto obtain tissue for pathological evaluation. Staging of breast cancerwould be improved by detecting new markers in cells, tissues, or bodilyfluids which could differentiate between different stages of invasion.Progress in this field will allow more rapid and reliable method fortreating breast cancer patients.

Treatment of breast cancer is generally decided after an accuratestaging of the primary tumor. Primary treatment options include breastconserving therapy (lumpectomy, breast irradiation, and surgical stagingof the axilla), and modified radical mastectomy. Additional treatmentsinclude chemotherapy, regional irradiation, and, in extreme cases,terminating estrogen production by ovarian ablation.

Until recently, the customary treatment for all breast cancer wasmastectomy. Fonseca et al., Annals of Internal Medicine 127:1013 (1997).However, recent data indicate that less radical procedures may beequally effective, in terms of survival, for early stage breast cancer.Fisher et al., J. of Clinical Oncology 16:441 (1998). The treatmentoptions for a patient with early stage breast cancer (i.e., stage Tis)may be breast-sparing surgery followed by localized radiation therapy atthe breast. Alternatively, mastectomy optionally coupled with radiationor breast reconstruction may be employed. These treatment methods areequally effective in the early stages of breast cancer.

Patients with stage I and stage II breast cancer requires surgery withchemotherapy and/or hormonal therapy. Surgery is of limited use in StageIII and stage IV patients. Thus, these patients are better candidatesfor chemotherapy and radiation therapy with surgery limited to biopsy topermit initial staging or subsequent restaging because cancer is rarelycurative at this stage of the disease. AJCC Cancer Staging Handbook 84,164-65 (Irvin D. Fleming et al. eds., 5^(th) ed. 1998).

In an effort to provide more treatment options to patients, efforts areunderway to define an earlier stage of breast cancer with low recurrencewhich could be treated with lumpectomy without postoperative radiationtreatment. While a number of attempts have been made to classify earlystage breast cancer, no consensus recommendation on postoperativeradiation treatment has been obtained from these studies. Page et al.,Cancer 75:1219 (1995); Fisher et al., Cancer 75:1223 (1995); Silversteinet al., Cancer 77:2267 (1996).

Ovarian Cancer

Cancer of the ovaries is the fourth-most common cause of cancer death inwomen in the United States, with more than 23,000 new cases and roughly14,000 deaths predicted for the year 2001. Shridhar, V. et al., CancerRes. 61(15): 5895-904 (2001); Memarzadeh, S. & Berek, J. S., J. Reprod.Med. 46(7): 621-29 (2001). The American Cancer Society estimates thatthere will be about 25,580 new cases of ovarian cancer in 2004 in theUnited States alone. Ovarian cancer will cause about 16,090 deaths inthe United States. ACS Website: cancer with the extension org of theworld wide web. The incidence of ovarian cancer is of serious concernworldwide, with an estimated 191,000 new cases predicted annually.Runnebaum, I. B. & Stickeler, E., J. Cancer Res. Clin Oncol. 127(2):73-79 (2001). Unfortunately, women with ovarian cancer are typicallyasymptomatic until the disease has metastasized Because effectivescreening for ovarian cancer is not available, roughly 70% of womendiagnosed have an advanced stage of the cancer with a five-year survivalrate of approximately 25-30%. Memarzadeh, S. & Berek, J. S., supra;Nunns, D. et al, Obstet. Gynecol. Surv. 55(12): 746-51. Conversely,women diagnosed with early stage ovarian cancer enjoy considerablyhigher survival rates. Werness, B. A. & Eltabbakh, G. H., Int'l J.Gynecol Pathol. 20(1): 48-63 (2001). Although our understanding of theetiology of ovarian cancer is incomplete, the results of extensiveresearch in this area point to a combination of age, genetics,reproductive, and dietary/environmental factors. Age is a key riskfactor in the development of ovarian cancer: while the risk fordeveloping ovarian cancer before the age of 30 is slim, the incidence ofovarian cancer rises linearly between ages 30 to 50, increasing at aslower rate thereafter, with the highest incidence being amongseptagenarian women. Jeanne M. Schilder et al., Hereditary OvarianCancer: Clinical Syndromes and Management, in Ovarian Cancer 182(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).

With respect to genetic factors, a family history of ovarian cancer isthe most significant risk factor in the development of the disease, withthat risk depending on the number of affected family members, the degreeof their relationship to the woman, and which particular first degreerelatives are affected by the disease. Id. Mutations in several geneshave been associated with ovarian cancer, including BRCA1 and BRCA2,both of which play a key role in the development of breast cancer, aswell as hMSH2 and hMLH1, both of which are associated with hereditarynon-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology,and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (StephenC. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCA1, located onchromosome 17, and BRCA2, located on chromosome 13, are tumor suppressorgenes implicated in DNA repair; mutations in these genes are linked toroughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at185-86. hMSH2 and hMLH1 are associated with DNA mismatch repair, and arelocated on chromosomes 2 and 3, respectively; it has been reported thatroughly 3% of hereditary ovarian carcinomas are due to mutations inthese genes. Look, supra at 173; Schilder et al., supra at 184, 188-89.

Reproductive factors have also been associated with an increased orreduced risk of ovarian cancer. Late menopause, nulliparity, and earlyage at menarche have all been linked with an elevated risk of ovariancancer. Schilder et al., supra at 182. One theory hypothesizes thatthese factors increase the number of ovulatory cycles over the course ofa woman's life, leading to “incessant ovulation,” which is thought to bethe primary cause of mutations to the ovarian epithelium. Id.; Laura J.Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic OvarianCancer, in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds.,2d ed. 2001). The mutations may be explained by the fact that ovulationresults in the destruction and repair of that epithelium, necessitatingincreased cell division, thereby increasing the possibility that anundetected mutation will occur. Id. Support for this theory may be foundin the fact that pregnancy, lactation, and the use of oralcontraceptives, all of which suppress ovulation, confer a protectiveeffect with respect to developing ovarian cancer. Id.

Among dietary/environmental factors, there would appear to be anassociation between high intake of animal fat or red meat and ovariancancer, while the antioxidant Vitamin A, which prevents free radicalformation and also assists in maintaining normal cellulardifferentiation, may offer a protective effect. Look, supra at 169.Reports have also associated asbestos and hydrous magnesium trisilicate(talc), the latter of which may be present in diaphragms and sanitarynapkins. Id. at 169-70.

Current screening procedures for ovarian cancer, while of some utility,are quite limited in their diagnostic ability, a problem that isparticularly acute at early stages of cancer progression when thedisease is typically symptomatic yet is most readily treated. Walter J.Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998);Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Werness &Eltabbakh, supra. Commonly used screening tests include biannualrectovaginal pelvic examination, radioimmunoassay to detect the CA-125serum tumor marker, and transvaginal ultrasonography. Burdette, supra at166.

Pelvic examination has failed to yield adequate numbers of earlydiagnoses, and the other methods are not sufficiently accurate. Id. Onestudy reported that only 15% of patients who suffered from ovariancancer were diagnosed with the disease at the time of their pelvicexamination. Look, supra at 174. Moreover, the CA-125 test is prone togiving false positives in pre-menopausal women and has been reported tobe of low predictive value in post-menopausal women. Id. at 174-75.Although transvaginal ultrasonography is now the preferred procedure forscreening for ovarian cancer, it is unable to distinguish reliablybetween benign and malignant tumors, and also cannot locate primaryperitoneal malignancies or ovarian cancer if the ovary size is normal.Schilder et al., supra at 194-95. While genetic testing for mutations ofthe BRCA1, BRCA2, hMSH2, and hMLH1 genes is now available, these testsmay be too costly for some patients and may also yield false negative orindeterminate results. Schilder et al., supra at 191-94.

Other markers of interest are HE4 and mesothelin. See Urban et al.Ovarian cancer screening Hematol Oncol Clin North Am. 2003 August;17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is a biomarkerfor ovarian carcinoma, Cancer Res. 2003 Jul. 1; 63(13):3695-700;Ordonez, Application of mesothelin immunostaining in tumor diagnosis, AmJ Surg Pathol. 2003 November; 27(11): 1418-28.

The staging of ovarian cancer, which is accomplished through surgicalexploration, is crucial in determining the course of treatment andmanagement of the disease. AJCC Cancer Staging Handbook 187 (Irvin D.Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh &Berek, supra; Shridhar et al., supra. Staging is performed by referenceto the classification system developed by the International Federationof Gynecology and Obstetrics. David H. Moore, Primary SurgicalManagement of Early Epithelial Ovarian Carcinoma, in Ovarian Cancer 203(Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al.eds., supra at 188. Stage I ovarian cancer is characterized by tumorgrowth that is limited to the ovaries and is comprised of threesubstages. Id. In substage IA, tumor growth is limited to one ovary,there is no tumor on the external surface of the ovary, the ovariancapsule is intact, and no malignant cells are present in ascites orperitoneal washings. Id. Substage IB is identical to IA, except thattumor growth is limited to both ovaries. Id. Substage IC refers to thepresence of tumor growth limited to one or both ovaries, and alsoincludes one or more of the following characteristics: capsule rupture,tumor growth on the surface of one or both ovaries, and malignant cellspresent in ascites or peritoneal washings. Id.

Stage II ovarian cancer refers to tumor growth involving one or bothovaries, along with pelvic extension. Id. Substage IIA involvesextension and/or implants on the uterus and/or fallopian tubes, with nomalignant cells in the ascites or peritoneal washings, while substageIIB involves extension into other pelvic organs and tissues, again withno malignant cells in the ascites or peritoneal washings. Id. SubstageIIC involves pelvic extension as in IIA or IIB, but with malignant cellsin the ascites or peritoneal washings. Id.

Stage III ovarian cancer involves tumor growth in one or both ovaries,with peritoneal metastasis beyond the pelvis confirmed by microscopeand/or metastasis in the regional lymph nodes. Id Substage IIIA ischaracterized by microscopic peritoneal metastasis outside the pelvis,with substage IIIB involving macroscopic peritoneal metastasis outsidethe pelvis 2 cm or less in greatest dimension. Id. Substage IIIC isidentical to IIIB, except that the metastasis is greater than 2 cm ingreatest dimension and may include regional lymph node metastasis. Id.Lastly, Stage IV refers to the presence distant metastasis, excludingperitoneal metastasis. Id.

While surgical staging is currently the benchmark for assessing themanagement and treatment of ovarian cancer, it suffers from considerabledrawbacks, including the invasiveness of the procedure, the potentialfor complications, as well as: the potential for inaccuracy. Moore,supra at 206-208, 213. In view of these limitations, attention hasturned to developing alternative staging methodologies throughunderstanding differential gene expression in various stages of ovariancancer and by obtaining various biomarkers to help better assess theprogression of the disease. Vartainen, J. et al., Int'l J. Cancer,95(5): 313-16 (2001); Shridhar et al. supra; Baekelandt, M. et al., J.Clin. Oncol. 18(22): 3775-81.

The treatment of ovarian cancer typically involves a multiprong attack,with surgical intervention serving as the foundation of treatment.Dennis S. Chi & William J. Hoskins, Primary Surgical Management ofAdvanced Epithlelial Ovarian Cancer, in Ovarian Cancer 241 (Stephen C.Rubin & Gregory P. Sutton eds., 2d ed. 2001). For example, in the caseof epithelial ovarian cancer, which accounts for approximately 90% ofcases of ovarian cancer, treatment typically consists of: (1)cytoreductive surgery, including total abdominal hysterectomy, bilateralsalpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2)adjuvant chemotherapy with paclitaxel and either cisplatin orcarboplatin. Eltabbakh, G. H. & Awtrey, C. S., Expert Op. Pharmacother.2(10): 109-24. Despite a clinical response rate of 80% to the adjuvanttherapy, most patients experience tumor recurrence within three years oftreatment. Id. Certain patients may undergo a second cytoreductivesurgery and/or second-line chemotherapy. Memarzadeh & Berek, supra.

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of ovarian cancer are of critical importance to the outcomeof the patient. Moreover, current procedures, while helpful in each ofthese analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop ovariancancer, for diagnosing ovarian cancer, for monitoring the progression ofthe disease, for staging the ovarian cancer, for determining whether theovarian cancer has metastasized, and for imaging the ovarian cancer.There is also a need for better treatment of ovarian cancer.

Pancreatic Cancer

Pancreatic cancer is the thirteenth-most common cancer and eighth-mostcause of cancer death worldwide. Donghui Li, Molecular Epidemiology, inPancreatic Cancer 3 (Douglas B. Evans et al. eds., 2002). In the UnitedStates, cancer of the pancreas is the fourth-most common cancer in bothmales and females, accounting for five percent of cancer deaths andnearly 30,000 deaths overall. Id. The rates of pancreatic cancer arehigher in men than women and higher in African-Americans as opposed toCaucasians. Id. at 9. The most significant predictor of pancreaticcancer is patient age; among Caucasians, the age-related incidence ofpancreatic cancer increases continuously, even through the 85 and oldercategory. Id. at 3. Approximately 80% of cases occur in the age range of60 to 80, with those in their 80 s experiencing a risk of acquiring thedisease 40 times that of those in their 40 s. Id. Furthermore, theAmerican Cancer Society estimates that there will be about 31,800 newcases of pancreatic cancer in 2004 in the United States alone.Pancreatic cancer will cause about 31,200 deaths in the United States inthe same year. ACS Website: cancer with the extension org of the worldwide web. Despite the efforts of researchers and physicians in devisingtreatments for pancreatic cancer, it remains almost universally fatal.James R. Howe, Molecular Markers as a Tool for the Early Diagnosis ofPancreatic Cancer, in Pancreatic Cancer 29 (Douglas B. Evans et al.eds., 2002).

Aside from age, a number of risk factors for pancreatic cancer have beenidentified, including smoking, diet, occupation, certain medicalconditions, heredity, and molecular biologic. Smoking is the mostimportant risk factor for acquiring the disease, with the link betweensmoking and pancreatic cancer being established in numerous studies. Li,supra at 3. The relative risk amounts to at least 1.5, increasing withthe level of smoking to an outer risk ratio of 10-fold. Id. The nextmost important factor would appear to be diet, with increased riskassociated with animal protein and fat intake, and decreased riskassociated with intake of fruits and vegetables. Id. at 3-4. As forparticular occupations, excessive rates of pancreatic cancer have beenassociated with workers in chemistry, coal and gas exploration, themetal industry, leather tanning, textiles, aluminum milling, andtransportation. Id. at 4. A number of medical conditions have also beenassociated with an increased incidence of pancreatic cancer, includingdiabetes, chronic pancreatitis, gastrectomy, and cholecystectomy,although the cause and effect relationship between these conditions andpancreatic cancer has not been established. Id.

Hereditary genetic factors comprise less than 10% of the pancreaticcancer burden, with associations documented with hereditarypancreatitis, as well as germline mutations in familial cancer syndromegenes such as hMSH2 and hMEH1 (hereditary nonpolyposis colon cancer),p16 (familial atypical multiple mole-melanoma) and BRCA1/BRCA2 (breastand ovarian cancer). Id. at 3. While no other organ has a higherinherited basis for cancer than the pancreas, researchers have beenunable to pinpoint the particular genetic defect(s) that contribute toone's susceptibility to pancreatic cancer. David H. Berger & William E.Fisher, Inherited Pancreatic Cancer Syndromes, in Pancreatic Cancer 73(Douglas B. Evans et al. eds., 2002).

From the standpoint of molecular biology, research has revealed anassociation between pancreatic cancer and a number of genetic mutations,including the activation of the proto-oncogene K-ras and theinactivation of the tumor suppressor genes p53, p16, and DPC4. Marina E.Jean et al., The Molecular Biology of pancreatic Cancer, in PancreaticCancer 15 (Douglas B. Evans et al. eds., 2002).

In one study of pancreatic adenocarcinomas, 83% possessed K-rasactivation along with inactivation of p16 and p53. Id. K-ras mutationsare found in 80 to 95% of pancreatic adenocarcinomas, with p53, p16, andDPC4 genes being the must frequently deleted tumor suppressor genes incancer of the pancreas. Howe, supra at 29. Homozygous deletions,hypermethylation, and mutations of the p16 gene have been discovered in85 to 98% of adenocarcinomas of the pancreas. Id. As might be expectedby the role of alterations in the K-ras, p53, p16, and DPC4 genes, lossof regulation of the cell cycle would appear to be key to tumorigenesisin the pancreas, and may explain why this cancer is so aggressive. Jean,supra at 15. Research has also revealed a link between this cancer andabnormal regulation of certain growth factors and growth factorreceptors, as well as an upregulation of matrix metalloproteinases andtumor angiogenesis regulators. Id. Epidermal growth factor, fibroblastgrowth factor, transforming growth factor-β, insulin-like growth factor,hepatocyte growth factor, and vascular endothelial growth factor mayplay various roles in pancreatic cancer, although such roles have notbeen elucidated. Id. at 18-22.

The development of screening techniques to detect the presence ofpancreatic cancer is particularly essential for this deadly cancer, asmost patients fail to present until their pancreatic tumors obstruct thebile duct or induce pain, at which point the tumors have invaded thecapillary and lymphatic vessels that surround the pancreas, Howe, supraat 29; unfortunately, patients with the metastatic form of the diseasetypically survive less than one year after diagnosis, Jean et al., supraat 15. While computed tomography (CT) and endoscopic retrogradecholangiopancreatography (ERCP) may assist in the diagnosis ofsymptomatic patients, there is presently no tool for screening forpancreatic tumors that would permit their early discovery, at whichpoint they might be curable. Howe, supra at 29. Markers such ascarcinoembryonic antigen, and antibodies generated against cell lines ofhuman colonic cancer (CA 19-9 and CA 195), human ovarian cancer (CA125), and human pancreatic cancer (SPAN-1 and DUPAN-2) may be elevatedin the serum of patients with pancreatic cancer, but these markers arenot sufficiently reliable to serve as screening tools due to their lackof specificity and appearance late in the disease. Walter J. Burdette,Cancer: Etiology, Diagnosis, and Treatment 99 (1998); Hasholzner, U. etal., Anticancer Res. 19(4A): 2477-80 (1999).

Due to the present lack of adequate screening methods, physicians areincreasingly turning to techniques which employ methods of molecularbiology as the most promising means for early diagnosis of the disease.Howe, supra at 30. At present, there is no high sensitivity, highspecificity marker that enables the detection of pancreatic cancer inasymptomatic individuals, but several biological markers are underinvestigation. Id. Considerable efforts are currently focusing on K-ras,with researchers devising techniques to screen samples of pancreaticjuice, bile, duodenal juice, or ERCP brushings to detect K-rasmutations. Id. Because the collection of these samples is invasive andnot particularly helpful in screening those who are asymptomatic,researchers have also turned to serum and stool analysis for K-rasmutations, with the former being the most promising, as the latter ishindered by the complexity of the source material. Id. at 35-38, 42.Moreover, because serum levels of the transcription factor protein p53may parallel cancer progression, p53 is likewise being studied aspossible tumor marker. Id. at 37; Jean et al., supra at 17.

Once pancreatic cancer has been diagnosed, treatment decisions are madein reference to the stage of cancer progression. A number of imagingtechniques are employed to stage pancreatic cancer, with computedtomography (CT) being the present method of choice, Harmeet Kaur et al.,Pancreatic Cancer: Radiologic Staging, in Pancreatic Cancer 86 (DouglasB. Evans et al. eds., 2002); Ishiguchi, T. et al, Hepatogastroenterology48(40): 923-27 (2001), despite the fact that it frequentlyunderestimates the extent of the cancer, as small-volume metastases areoften beyond the resolution of CT, H. J. Kim & K. C. Conlon,Laparascopic Staging, in Pancreatic Cancer 15 (Douglas B. Evans et al.eds., 2002). MRI may at some point supplant CT in view of, inter alia,its ability to (1) contrast among various tissue, (2) modify pulsesequences to improve visualization of lesions and minimize artifacts,(3) perform imaging while limiting a patient's exposure to ionizingradiation, and (4) visualize vessels without using IV iodinated contrastreagents. Kaur et al., supra at 87. At present, however, MRI has notdemonstrated a clear advantage over CT. Kim & Conlon, supra at 116.

A variety of ultrasonic techniques are also currently employed instaging, including transabdominal ultrasound (TUS), endoscopicultrasound (EUS), and intraoperative ultrasound (IUS), with EUS beingone of the most promising. Kaur et al., supra at 86; Richard A.Erickson, Endoscopic Diagnosis and Staging: Endoscopic Ultrasound,Endoscopic Retrograde Cholangiopancreatography, in Pancreatic Cancer97-106 (Douglas B. Evans et al. eds., 2002). These techniques, however,are each limited by a variety of factors: TUS is hindered by gas in thegastrointestinal tract and fat in the peritoneum, EUS requiresconsiderable experience in ultrasonography and endoscopy and may not bewidely available, and IUS can only be used intraoperatively. Kaur etal., supra at 86.

Although in its nascent stages, the search for markers that will assistin staging pancreatic cancer has found some possible leads. For example,research has revealed that two metastasis-suppressing genes, nm23-H1 andKAI1, are differentially expressed depending on the stage of pancreaticcancer, with their expression being upregulated at early stages and downregulated at later stages of the disease. Friess, H. et al., J. Clin.Oncol. 19(9): 2422-32 (2001). Researchers have also focused on geneticlymph node staging, particularly searching for mutations in the K-rasproto-oncogene. Yamada, T. et al., Int'l J. Oncol. 16(6): 1165-71(2000). Likewise, research has identified that the presence of mutatedK-ras sequences in plasma/serum is associated with late stage pancreaticcancer, although the presence of early stage pancreatic cancer can bedetected this way as well. Sorenson, G. D., Clin. Cancer Res. 6(6):2129-37 (2000). A promising staging technique using a multimarkerreverse transcriptase-polymerase chain reaction assay has successfullydistinguished pancreatic cancer stages by assaying blood and tissuesamples for mRNA expression of the following tumor markers: the β-humanchorionic gonadotropin gene, the hepatocyte growth factor receptor genec-met, and the β-1,4-N-acetyl-galactosaminyl-transferase gene. Bilchik,A. et al., Cancer 88(5): 1037-44 (2000).

One classification system commonly used to stage pancreatic cancer isthe TNM system devised by the Union Internationale Contre le Cancer.AJCC Cancer Staging Handbook 3 (Irvin D. Fleming et al. eds., 5^(th) ed.1998). This system is divided into several stages, each of whichevaluates the extent of cancer growth with respect to primary tumor (T),regional lymph nodes (N), and distant metastasis (M). Id.

Stage 0 is characterized by carcinoma in situ (Tis), with no regionallymph node metastasis (N0) and no distant metastasis (M0). Id. at 113.Stages I and II differ from stage 0 only in terms of tumor category:stage I involves a tumor limited only to the pancreas that is either (1)2 cm or less in greatest dimension (T1) or (2) more than 2 cm ingreatest dimension (T2), while stage II involves a tumor that extendsdirectly into the duodenum, bile duct, or peripancreatic tissues (T3).Id. Stage III involves tumor category T1, T2, or T3; regional lymph nodemetastasis (N1), which involves either a single lymph node (pN1a) ormultiple lymph nodes (pN1b); and no distant metastasis (M0). Stage IVAis characterized by tumor extension directly into the stomach, spleen,colon, or adjacent large vessels (T4); any N category; and no distantmetastasis (M0). Lastly, stage IVB is characterized by any T category,any N category, and distant metastasis (M1). Id.

Once the cancer has been staged, the only consistently effectivetreatment for the disease is surgery, and with only ten to fifteenpercent of patients being able to undergo potentially curativeresection. Jean et al., supra at 15; Fleming et al. eds., supra at 111;William F. Regine, Postoperative Adjuvant Therapy: Past, Present, andFuture Trial Development, in Pancreatic Cancer 235 (Douglas B. Evans etal eds., 2002). Moreover, the five-year survival of those patientsundergoing resection is below twenty percent. Regine, supra at 235.While chemotherapeutic agents such as gemcitabine and 5-fluorouracilhave shown some effectiveness against pancreatic carcinomas, the realityis that chemotherapy has shown little impact on survival from pancreaticcancer. Burdette, supra at 101. Radiation therapy has providedconflicting results with respect to its efficacy, id., althoughradiation in combination with 5-fluorouracil has shown some promise,Regine, supra at 235.

In view of the failure of conventional techniques at treating pancreaticcancer, a number of novel approaches employing the techniques ofmolecular biology have been investigated. Considerable research has beenperformed in the area of gene therapy, including antisense technology,gene-directed prodrug activation strategies, promoter gene strategies,and oncolytic viral therapies. Eugene A. Choi & Francis R. Spitz,Strategies for Gene Therapy, in Pancreatic Cancer 331 (Douglas B. Evanset al. eds., 2002); Kasuya, H. et al, Hepatogastroenterology 48(40):957-61 (2001). Other recent approaches have focused on the inhibition ofmatrix metalloproteinases, enzymes which facilitate the metastasis andinvasion of tumor cells through their degradation of basement membranes,and their role in peritumoral stromal degradation and angiogenesis.Alexander S. Rosemurgy, II & Mahmudul Haq, Role of MatrixMetalloproteinase Inhibition in the Treatment of Pancreatic Cancer, inPancreatic Cancer 369 (Douglas B. Evans et al. eds., 2002).

From the foregoing, it is clear that procedures used for detecting,diagnosing, monitoring, staging, prognosticating, and preventing therecurrence of pancreatic cancer are of critical importance to theoutcome of the patient. Moreover, current procedures, while helpful ineach of these analyses, are limited by their specificity, sensitivity,invasiveness, and/or their cost. As such, highly specific and sensitiveprocedures that would operate by way of detecting novel markers incells, tissues, or bodily fluids, with minimal invasiveness and at areasonable cost, would be highly desirable.

Lung Cancer

Throughout the last hundred years, the incidence of lung cancer hassteadily increased, so much so that now in many countries, it is themost common cancer. In fact lung cancer is the second most prevalenttype of cancer for both men and women in the United States and is themost common cause of cancer death in both sexes. Lung cancer deaths haveincreased ten-fold in both men and women since 1930, primarily due to anincrease in cigarette smoking, but also due to an increased exposure toarsenic, asbestos, chromates, chloromethyl ethers, nickel, polycyclicaromatic hydrocarbons and other agents. See Scott, Lung Cancer: A Guideto Diagnosis and Treatment, Addicus Books (2000) and Alberg et al., inKane et al. (eds.) Biology of Lung Cancer, pp. 11-52, Marcel Dekker,Inc. (1998). The American Cancer Society estimates there will be over173,550 new cases of lung cancer in 2004. Additionally, there will be anestimated 160,440 deaths from lung cancer in 2004. ACS Website: cancerwith the extension org of the world wide web.

Lung cancer may result from a primary tumor originating in the lung or asecondary tumor which has spread from another organ such as the bowel orbreast Although there are over a dozen types of lung cancer, over 90%fall into two categories: small cell lung cancer (SCLC) and non-smallcell lung cancer (NSCLC). See Scott, supra. About 20-25% of all lungcancers are characterized as SCLC, while 70-80% are diagnosed as NSCLC.Id. A rare type of lung cancer is mesothelioma, which is generallycaused by exposure to asbestos, and which affects the pleura of thelung. Lung cancer is usually diagnosed or screened for by chest x-ray,CAT scans, PET scans, or by sputum cytology. A diagnosis of lung canceris usually confirmed by biopsy of the tissue. Id.

SCLC tumors are highly metastatic and grow quickly. By the time apatient has been diagnosed with SCLC, the cancer has usually alreadyspread to other parts of the body, including lymph nodes, adrenals,liver, bone, brain and bone marrow. See Scott supra; Van Houtte et al.(eds.), Progress and Perspective in the Treatment of Lung Cancer,Springer-Verlag (1999). Because the disease has usually spread to suchan extent that surgery is not an option, the current treatment of choiceis chemotherapy plus chest irradiation. See Van Houtte, supra. The stageof disease is a principal predictor of long-term survival. Less than 5%of patients with extensive disease that has spread beyond one lung andsurrounding lymph nodes, live longer than two years. Id. However, theprobability of five-year survival is three to four times higher if thedisease is diagnosed and treated when it is still in a limited stage,i.e., not having spread beyond one lung. Id.

NSCLC is generally divided into three types: squamous cell carcinoma,adenocarcinoma and large cell carcinoma. Both squamous cell cancer andadenocarcinoma develop from the cells that line the airways; however,adenocarcinoma develops from the goblet cells that produce mucus. Largecell lung cancer has been thus named because the cells look large androunded when viewed microscopically, and generally are consideredrelatively undifferentiated. See Yesner, Atlas of Lung Cancer,Lippincott-Raven (1998).

Secondary lung cancer is a cancer initiated elsewhere in the body thathas spread to the lungs. Cancers that metastasize to the lung include,but are not limited to, breast cancer, melanoma, colon cancer andHodgkin's lymphoma Treatment for secondary lung cancer may depend uponthe source of the original cancer. In other words, a lung cancer thatoriginated from breast cancer may be more responsive to breast cancertreatments and a lung cancer that originated from the colon cancer maybe more responsive to colon cancer treatments.

The stage of a cancer indicates how far it has spread and is animportant indicator of the prognosis. In addition, staging is importantbecause treatment is often decided according to the stage of a cancer.SCLC is divided into two stages: limited disease, i.e., cancer that canonly be seen in one lung and in nearby lymph nodes; and extensivedisease, i.e., cancer that has spread outside the lung to the chest orto other parts of the body. For most patients with SCLC, the disease hasalready progressed to lymph nodes or elsewhere in the body at the timeof diagnosis. See Scott, supra. Even if spreading is not apparent on thescans, it is likely that some cancer cells may have spread away andtraveled through the bloodstream or lymph system. In general,chemotherapy with or without radiotherapy is often the preferredtreatment. The initial scans and tests done at first will be used laterto see how well a patient is responding to treatment.

In contrast, non-small cell cancer may be divided into four stages.Stage I is highly localized cancer with no cancer in the lymph nodes.Stage II cancer has spread to the lymph nodes at the top of the affectedlung. Stage in cancer has spread near to where the cancer started. Thiscan be to the chest wall, the covering of the lung (pleura), the middleof the chest (mediastinum) or other lymph nodes. Stage IV cancer hasspread to another part of the body. Stage I-III cancer is usuallytreated with surgery, with or without chemotherapy. Stage IV cancer isusually treated with chemotherapy and/or palliative care.

A number of chromosomal and genetic abnormalities have been observed inlung cancer. In NSCLC, chromosomal aberrations have been described on3p, 9p, 11p, 15p and 17p, and chromosomal deletions have been seen onchromosomes 7, 11, 13 and 19. See Skarin (ed.), Multimodality Treatmentof Lung Cancer, Marcel Dekker, Inc. (2000); Gemmill et al., pp. 465-502,in Kane, supra; Bailey-Wilson et al., pp. 53-98, in Kane, supra.Chromosomal abnormalities have been described on 1p, 3p, 5q, 6q, 8q, 13qand 17p in SCLC. Id. In addition, the loss of the short arm ofchromosome 3p has also been seen in greater than 90% of SCLC tumors andapproximately 50% of NSCLC tumors. Id.

A number of oncogenes and tumor suppressor genes have been implicated inlung cancer. See Mabry, pp. 391-412, in Kane, supra and Sclafani et al.,pp. 295-316, in Kane, supra. In both SCLC and NSCLC, the p53 tumorsuppressor gene is mutated in over 50% of lung cancers. See Yesner,supra. Another tumor suppressor gene, FHIT, which is found on chromosome3p, is mutated by tobacco smoke. Id.; Skarin, supra. In addition, morethan 95% of SCLCs and approximately 20-60% of NSCLCs have an absent orabnormal retinoblastoma (Rb) protein, another tumor suppressor gene. Theras oncogene (particularly K-ras) is mutated in 20-30% of NSCLCspecimens and the c-erbB2 oncogene is expressed in 18% of stage 2 NSCLCand 60% of stage 4 NSCLC specimens. See Van Houtte, supra. Other tumorsuppressor genes that are found in a region of chromosome 9,specifically in the region of 9p21, are deleted in many cancer cells,including p16^(INK4A) and p15^(INK4B). See Bailey-Wilson, supra;Sclafani et al., supra. These tumor suppressor genes may also beimplicated in lung cancer pathogenesis.

In addition, many lung cancer cells produce growth factors that may actin an autocrine or paracrine fashion on lung cancer cells. See Siegfriedet al., pp. 317-336, in Kane, supra, Moody, pp. 337-370, in Kane, supraand Heasley et al., 371-390, in Kane, supra. In SCLC, many tumor cellsproduce gastrin-releasing peptide (GRP), which is a proliferative growthfactor for these cells. See Skarin, supra. Many NSCLC tumors expressepidermal growth factor (EGF) receptors, allowing NSCLC cells toproliferate in response to EGF. Insulin-like growth factor (IGF-1) iselevated in greater than 95% of SCLC and greater than 80% of NSCLCtumors; it is thought to function as an autocrine growth factor. Id.Finally, stem cell factor (SCF, also known as steel factor or kitligand) and c-Kit (a proto-oncoprotein tyrosine kinase receptor for SCF)are both expressed at high levels in SCLC, and thus may form anautocrine loop that increases proliferation. Id.

Although the majority of lung cancer cases are attributable to cigarettesmoking, most smokers do not develop lung cancer. Epidemiologicalevidence has suggested that susceptibility to lung cancer may beinherited in a Mendelian fashion, and thus have an inherited geneticcomponent. Bailey-Wilson, supra. Thus, it is thought that certainallelic variants at some genetic loci may affect susceptibility to lungcancer. Id. One way to identify which allelic variants are likely to beinvolved in lung cancer susceptibility, as well as susceptibility toother diseases, is to look at allelic variants of genes that are highlyexpressed in lung.

The lung is susceptible to a number of other debilitating diseases aswell, including, without limitation, emphysema, pneumonia, cysticfibrosis and asthma. See Stockley (ed.), Molecular Biology of the Lung,Volume I: Emphysema and Infection, Birkhauser Verlag (1999), hereafterStockley I, and Stockley (ed.), Molecular Biology of the Lung, VolumeII: Asthma and Cancer, Birkhauser Verlag (1999), hereafter Stockley II.The cause of many these disorders is still not well understood and thereare few, if any, good treatment options for many of these noncancerouslung disorders. Thus, there remains a need to understand variousnoncancerous lung disorders and to identify treatments for thesediseases.

The development and differentiation of lung tissue during embryonicdevelopment is also very important. All of the epithelial cells of therespiratory tract, including those of the lung and bronchi, are derivedfrom the primitive endodermal cells that line the embryonic outpouching.See Yesner, supra. During embryonic development, multipotent endodermalstem cells differentiate into many different types of specialized cells,which include ciliated cells for moving inhaled particles, goblet cellsfor producing mucus, Kulchitsky's cells for endocrine function, andClara cells and type II pneumocytes for secreting surfactant protein.Id. Improper development and differentiation may cause respiratorydisorders and distress in infants, particularly in premature infants,whose lungs cannot produce sufficient surfactant when they are born.Further, some lung cancer cells, particularly small cell carcinomas, areplastic and can alter their phenotype into a number of cell types,including large cell carcinoma, adenocarcinoma and squamous cellcarcinoma. Id. Thus, a better understanding of lung development anddifferentiation may help facilitate understanding of lung cancerinitiation and progression.

The most common screening tests for lung cancer are chest x-ray andsputum cytology. Randomized controlled trials have not demonstrated areduction in lung cancer mortality resulting from screening with chestx-ray and/or sputum cytology. Additionally, sputum cytology has not beenshown to be effective when used as an adjunct to annual chest x-ray.Screening with chest x-ray plus sputum cytology appears to detect lungcancer at an earlier stage, but this would be expected in a screeningtest whether or not it was effective at reducing mortality. Since earlydetection by current screening methods fails to reduce mortality in lungcancer patients, current lung cancer screening methods are inadequate.

There are two important potential hazards associated with chestradiography screening. First, false positive test results can lead to anunnecessary invasive procedure, such as percutaneous needle biopsy orthoracotomy. These procedures are costly and due to their invasivenature carry risks of their own. The second hazard with chestradiography screening is overdiagnosis. Overdiagnosis is the diagnosisof a small or slowly growing tumor that would not have become clinicallysignificant had it not been defected by screening. Althoughoverdiagnosis is almost impossible to document in a living individual,autopsy studies suggest that many individuals die with lung cancerrather than from it.

Additionally, the spectrum of lung cancer type has shifted over the lasttwo decades. Whereas the most common type used to be squamous cellcancer (usually centrally located), the most common type now isadenocarcinoma (usually peripherally located). The latter may be moreamenable to early detection by chest x-ray, the limitations of which aredescribed above. In contrast, sputum cytology, is more sensitive in thedetection of squamous cell cancer than in detecting adenocarcinoma, andtherefore lacks usefulness in detecting the more common adenocarcinomas.Clearly, new highly sensitive non-invasive methods of detecting lungcancer are needed.

There are intensive efforts to improve lung cancer screening with newertechnologies, including low-dose helical computed tomography (LDCT) andmolecular techniques. LDCT is far more sensitive than chest radiography.In a recent screening study, CT detected almost 6 times as many stage Ilung cancers as chest radiography and most of these tumors were 1 cm orless in diameter. However, the effectiveness of screening with LDCT hasnot yet been evaluated in a controlled clinical trial.

There are two potential hazards that must be considered against anypotential benefit of screening with LDCT. The more common and familiarhazard is the false positive test result, which may lead to anxiety andinvasive diagnostic procedures. A less familiar hazard is overdiagnosis,the diagnosis of a condition that would not have become clinicallysignificant had it not been detected by screening. In the case ofscreening with LDCT, overdiagnosis could lead to unnecessary diagnosisof lung cancer requiring some combination of surgery, e.g., lobectomy,chemotherapy and radiation therapy. As stated above, overdiagnosis isalmost impossible to document in a living individual. In one largestudy, about one-sixth of all lung cancers found at autopsy had not beenclinically recognized before death. Furthermore, autopsy probably failsto detect many small lung cancers that are detectable by CT.

Current therapies for lung cancer are quite limited. Generally, patientoptions comprise surgery, radiation therapy, and chemotherapy.

Depending on the type and stage of a lung cancer, surgery may be used toremove the tumor along with some surrounding lung tissue. A lobectomyrefers to a lobe (section) of the lung being removed. If the entire lungis removed, the surgery is called a pneumonectomy. Removing only part ofa lobe is known as a segmentectomy or wedge resection.

If the cancer has spread to the brain, benefit may be gained fromremoval of the brain metastasis. This involves a craniotomy (surgerythrough a hole in the skull).

For radiation therapy several methods exist. External beam radiationtherapy uses radiation delivered from outside the body that is focusedon the cancer. This type of radiation therapy is most often used totreat a primary lung cancer or its metastases to other organs.

Brachytherapy uses a small pellet of radioactive material placeddirectly into the cancerous tissue or into the airway next to thecancer. Radiation therapy is sometimes used as the main (primary)treatment of lung cancer, especially if the general health of thepatient is too poor to undergo surgery. Brachytherapy can also be usedto help relieve blockage of large airways by cancer.

Additionally, radiation therapy can be used as a post surgical treatmentto kill very small deposits of cancer that cannot be seen or removedduring surgery. Radiation therapy can also be used to palliate (relieve)symptoms of lung cancer such as pain, bleeding, difficulty swallowing,and problems caused by brain metastases.

For chemotherapy, cisplatin or a related drug, carboplatin, are thechemotherapy agents most often used in treating NSCLC. Recent studiesfound that combining either of these with drugs such as gemcitabine,paclitaxel, docetaxel, etoposide, or vinorelbine appear to be moreeffective in treating NSCLC.

Recently, the National Comprehensive Cancer Network (NCCN; nccn with theextension org of the world wide web), an alliance of nineteen of theworld's leading cancer centers, announces a major update of the NCCNNon-Small Cell Lung Cancer Clinical Practice Guidelines. The NCCN iswidely recognized as a standard for clinical policy in oncology.

Recently approved targeted therapy, gefitinib (Iressa®, AstraZenecaPharmaceuticals LP) is now recommended as third-line therapy and assecond-line only if the platinum/docetaxel combination was used asfirst-line therapy.

The NCCN's Non-Small Cell Lung Cancer (NSCLC) guidelines containrecommendations for administration of chemotherapy to patients with thisdisease including patient selection criteria and definition of first-,second-, and third-line agents and combinations.

Chemotherapeutic agents are specified as two-agent regimens forfirst-line therapy, two agent regimens or single agents for second-linetherapy, and one single agent for third-line therapy. Agents used infirst- and second-line therapy are: cisplatin (Platinol®, Bristol-MyersSquibb Company), carboplatin (Paraplatin®, Bristol-Myers SquibbCompany), paclitaxel (Taxol®, Bristol-Myers Squibb Company), docetaxel(Taxotere®, Aventis Pharmaceuticals Inc.), vinorelbine (Navelbine®,GlaxoSmithKline), gemcitabine (Gemzar®, Eli Lilly and Company),etoposide (Toposar®, Pfizer, Inc.; VePesid®, Bristol-Myers SquibbCompany; Etopophos®, Bristol-Myers Squibb Company), irinotecan(Camptosar®, Pfizer, Inc.), vinblastine (Velban®, Eli Lilly andCompany), mitomycin (Mutamycin®, Bristol-Myers Squibb Company), andifosfamide (Ifex®, Bristol-Myers Squibb Company).

Some of the usual chemotherapy combinations used for patients with SCLCinclude: EP (etoposide and cisplatin); ET (etoposide and carboplatin);ICE (ifosfamide, carboplatin, and etoposide); and CAV (cyclophosphamide,doxorubicin, and vincristine).

New drugs such as gemcitabine, paclitaxel, vinorelbine, topotecan, andteniposide have shown promising results in some SCLC studies. Growthfactors may be given in conjunction to chemotherapy agents if patienthealth is good. The administration of growth factors help prevent bonemarrow side effects.

Ongoing or recently completed therapeutic trials for various compoundsto treat lung cancer include alitretinoin (Panretin®, LigandPharmaceuticals), topotecan HCl (Hycamtin® GlaxoSmithKline), liposomalether lipid (Elan Pharmaceutical), cantuzumab mertansine (ImmunoGen),Gavax® (Cell Genesys), vincristine (Onco TCS®, Inex Pharmaceuticals),Neovastat® (AEterna Laboratories), squalamine (Genaera), mirostipen(Human Genome Sciences Inc.), Advexin® (Introgen Therapeutics),biricodar dicitrate (Incel®, Vertex Pharmaceuticals), flavopiridol(Aventis), Affintac® (Eli Lilly and Company), pivaloyloxymethylbutyrate(Pivanex®, Titan Pharmaceuticals), tirapazamine (Tirazone®,Sanofi-Synthelabo Pharmaceuticals), irinotecan (Camptosar®, Pharmacia),tezacitabine (Chiron), cisplatin/vinblastine/amifostine (MedImmune),paclitaxel/carboplatin/amifostine (MedImmune), Oncomyc-NG® (AVIBioPharma), exisulind/vinorelbine (Aptosyn)/Navelbine®, Cell Pathyways),tariquidar (QLIT), Xyotax® (Cell Therapeutics), PEG-camptothecin(Prothecan®, Enzon), decitabine (SuperGen), Tarceva® (OSIPharmaceuticals), ABX-EGF (Abgenix), Tocosol Paclitaxel® (SonusPharmaceuticals), TheraFab® (Antisoma), minodronate (YamanouchiPharmaceutical), exisulind/docetaxel/carboplatin(Aptosyn®/Taxotere®/Paraplatin®, Cell Pathways), exisulind/gemcitabineHCl (Aptosyn®/Gemzar®, Cell Pathways), IMC-C225/carboplatin/paclitaxel(Erbitux®/carboplatin®/paclitaxel®, ImClone Systems), and vinorelbine(Navelbine®, GlaxoSmithKline).

As indicated above, many therapeutics are recommended for use incombination as a first-line therapy or only if other therapeutics havefailed as second-, and third-line agents. While there are many compoundsin ongoing or recently completed therapeutic trials, there is great needfor additional therapeutic compounds capable of treating early stage andadvanced or metastasized lung cancer.

Accordingly, there is a great need for more sensitive and accuratemethods for predicting whether a person is likely to develop lungcancer, for diagnosing lung cancer, for monitoring the progression ofthe disease, for staging the lung cancer, for determining whether thelung cancer has metastasized and for imaging the lung cancer. There isalso a need for better treatment of lung cancer. Further, there is agreat need for diagnosing and treating noncancerous lung disorders suchas emphysema, pneumonia, lung infection, pulmonary fibrosis, cysticfibrosis and asthma. There is also a need for compositions and methodsof using these compositions to identify lung tissue for forensicpurposes and for determining whether a particular cell or tissueexhibits lung-specific characteristics.

As discussed above, each of the methods for diagnosing and stagingbreast, ovarian pancreatic, and lung cancer is limited by the technologyemployed. Accordingly, there is need for sensitive molecular andcellular markers and reagents for the detection of breast, ovarian,pancreatic and lung cancer including metastatic cancer. There is a needfor molecular markers and reagents for the accurate staging, includingclinical and pathological staging, of breast, ovarian, pancreatic andlung cancers to optimize treatment methods. Finally, there is a need forsensitive molecular and cellular markers and reagents to monitor theprogress of cancer treatments, including markers that can detectrecurrence of breast, ovarian, pancreatic and lung cancers followingremission.

The present invention provides alternative reagents and methods fortreating breast, ovarian, pancreatic and lung cancer that overcome thelimitations of conventional therapeutic methods as well as offeradditional advantages that will be apparent from the detaileddescription below.

Angiogenesis in Cancer

Growth and metastasis of solid tumors are also dependent onangiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473; Folkman,J., 1989, Journal of the National Cancer Institute, 82, 4-6. It has beenshown, for example, that tumors which enlarge to greater than 2 mm mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. Once these new blood vessels become embedded inthe tumor, they provide a means for tumor cells to enter the circulationand metastasize to distant sites such as liver, lung or bone. Weidner,N., et al., 1991, The New England Journal of Medicine, 324(1), 1-8.

Angiogenesis, defined as the growth or sprouting of new blood vesselsfrom existing vessels, is a complex process that primarily occurs duringembryonic development. The process is distinct from vasculogenesis, inthat the new endothelial cells lining the vessel arise fromproliferation of existing cells, rather than differentiating from stemcells. The process is invasive and dependent upon proteolysis of theextracellular matrix (ECM), migration of new endothelial cells, andsynthesis of new matrix components. Angiogenesis occurs duringembryogenic development of the circulatory system; however, in adulthumans, angiogenesis only occurs as a response to a pathologicalcondition (except during the reproductive cycle in women).

Under normal physiological conditions in adults, angiogenesis takesplace only in very restricted situations such as hair growth andwounding healing. Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther.63(3):265-311; Ribatti et al., 1991, Haematologica 76(4):3 11-20; Risau,1997, Nature 386(6626):67 1-4. Angiogenesis progresses by a stimuluswhich results in the formation of a migrating column of endothelialcells. Proteolytic activity is focused at the advancing tip of this“vascular sprout”, which breaks down the ECM sufficiently to permit thecolumn of cells to infiltrate and migrate. Behind the advancing front,the endothelial cells differentiate and begin to adhere to each other,thus forming a new basement membrane. The cells then cease proliferationand finally define a lumen for the new arteriole or capillary.

Unregulated angiogenesis has gradually been recognized to be responsiblefor a wide range of disorders, including, but not limited to, cancer,cardiovascular disease, rheumatoid arthritis, psoriasis and diabeticretinopathy. Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999,Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2):158-64.

Of particular interest is the observation that angiogenesis is requiredby solid tumors for their growth and metastases. Folkman, 1986 supra;Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6; Folkman, 1992, SeminCancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24. A tumorusually begins as a single aberrant cell which can proliferate only to asize of a few cubic millimeters due to the distance from availablecapillary beds, and it can stay ‘dormant’ without further growth anddissemination for a long period of time. Some tumor cells then switch tothe angiogenic phenotype to activate endothelial cells, whichproliferate and mature into new capillary blood vessels. These newlyformed blood vessels not only allow for continued growth of the primarytumor, but also for the dissemination and recolonization of metastatictumor cells. The precise mechanisms that control the angiogenic switchis not well understood, but it is believed that neovascularization oftumor mass results from the net balance of a multitude of angiogenesisstimulators and inhibitors Folkman, 1995, supra.

One of the most potent angiogenesis inhibitors is endostatin identifiedby O'Reilly and Folkman. O'Reilly et al., 1997, Cell 88(2):277-85;O'Reilly et al., 1994, Cell 79(2):3 15-28. Its discovery was based onthe phenomenon that certain primary tumors can inhibit the growth ofdistant metastases. O'Reilly and Folkman hypothesized that a primarytumor initiates angiogenesis by generating angiogenic stimulators inexcess of inhibitors. However, angiogenic inhibitors, by virtue of theirlonger half-life in the circulation, reach the site of a secondary tumorin excess of the stimulators. The net result is the growth of primarytumor and inhibition of secondary tumor. Endostatin is one of a growinglist of such angiogenesis inhibitors produced by primary tumors. It is aproteolytic fragment of a larger protein: endostatin is a 20 kDafragment of collagen XVIII (amino acid H1132-K1315 in murine collagenXVIII). Endostatin has been shown to specifically inhibit endothelialcell proliferation in vitro and block angiogenesis in vivo. Moreimportantly, administration of endostatin to tumor-bearing mice leads tosignificant tumor regression, and no toxicity or drug resistance hasbeen observed even after multiple treatment cycles. Boehm et al., 1997,Nature 390(6658):404-407. The fact that endostatin targets geneticallystable endothelial cells and inhibits a variety of solid tumors makes ita very attractive candidate for anticancer therapy. Fidler and Ellis,1994, Cell 79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84;Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition,angiogenesis inhibitors have been shown to be more effective whencombined with radiation and chemotherapeutic agents. Klement, 2000, J.Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,Nature 394(6690):287-91.

The present invention provides alternative methods of treating breast,ovarian, pancreatic and lung cancer that overcome the limitations ofconventional therapeutic methods as well as offer additional advantagesthat will be apparent from the detailed description below.

SUMMARY OF THE INVENTION

This invention is directed to an isolated Pro104 antibody that binds toPro104 on a mammalian cell in vivo. The invention is further directed toan isolated Pro104 antibody that internalizes upon binding to Pro104 ona mammalian cell in vivo. The antibody may be a monoclonal antibody.Alternatively, the antibody is an antibody fragment or a chimeric or ahumanized antibody. The monoclonal antibody may be produced by ahybridoma selected from the group of hybridomas deposited under AmericanType Culture Collection accession number PTA-5277, 6076, 6077 and 6078.

The antibody may compete for binding to the same epitope as the epitopebound by the monoclonal antibody produced by a hybridoma selected fromthe group of hybridomas deposited under the American Type CultureCollection accession number PTA-5277, 6076, 6077 and 6078.

The invention is also directed to conjugated antibodies. They may beconjugated to a growth inhibitory agent or a cytotoxic agent. Thecytotoxic agent may be selected from the group consisting of toxins,antibiotics, radioactive isotopes and nucleolytic enzymes and toxins.Examples of toxins include, but are not limited to, maytansin,maytansinoids, saporin, gelonin, ricin or calicheamicin.

The mammalian cell may be a cancer cell. Preferably, the anti-Pro104monoclonal antibody inhibits the growth of Pro104-expressing cancercells in vivo.

The antibody may be produced in bacteria. Alternatively, the antibodymay be a humanized form of an anti-Pro104 antibody produced by ahybridoma selected from the group of hybridomas having ATCC accessionnumber PTA-5277, 6076, 6077 and 6078.

Preferably, the cancer is selected from the group consisting of breast,ovarian, pancreatic and lung cancer. The invention is also directed to amethod of producing the antibodies comprising culturing an appropriatecell and recovering the antibody from the cell culture.

The invention is also directed to compositions comprising the antibodiesand a carrier. The antibody may be conjugated to a cytotoxic agent. Thecytotoxic agent may be a radioactive isotope or other chemotherapeuticagent.

The invention is also directed to a method of killing aPro104-expressing cancer cell, comprising contacting the cancer cellwith the antibodies of this invention, thereby killing the cancer cell.The cancer cell may be selected from the group consisting of breast,ovarian, pancreatic and lung cancer cells.

The ovarian cancer may be ovarian serous adenocarcinoma.

The breast cancer may be breast infiltrating ductal carcinoma.

The breast, ovarian, pancreatic or lung cancer may also be metastatic.

The invention is also directed to a method of alleviating aPro104-expressing cancer in a mammal, comprising administering atherapeutically effective amount of the antibodies to the mammal.

In addition, the invention is directed to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody as described herein. The article ofmanufacture may also comprise an additional component, e.g., a packageinsert indicating that the composition can be used to treat ovarian orpancreatic cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that Pro104.D116.1 MAb binds to 293F cells transientlytransfected with Pro104.

FIG. 2 shows that Pro104.D118.1 MAb Binds to 293F cells transientlytransfected with Pro104.

FIG. 3 shows that Pro104.C19.1 binds to live HeLa cancer cellsexpressing Pro104.

FIG. 4 shows that Cy3-Pro104.C25.1 binds to live HeLa cancer cellsexpressing Pro104.

FIG. 5 shows that Cy3-Pro104.C25.1 binds to and is internalized in liveHeLa cancer cells expressing Pro104.

FIG. 6 shows that Cy3-Pro104.C19.1 binds to and is internalized inpancreatic cancer cells expressing Pro104.

FIG. 7 shows that Cy3-Pro104.C55.1 binds to and is internalized inpancreatic cancer cells expressing Pro104.

FIG. 8 shows that Pro104.C25.1 binds to Pro104 on cancer cells inovarian tumors.

FIG. 9 shows that Pro104.C25.1 binds to Pro104 on the cell membrane ofovarian cancer cells.

FIG. 10 shows that Pro104.D9 binds to Pro104 on the cell membrane ofovarian cancer cells.

FIG. 11 shows that Pro104.D133 binds to Pro104 on the cell membrane ofserous ovarian cancer cells.

FIG. 12 shows that Pro104.C25.1 binds to Pro104 on cancer cells inpancreatic tumors.

FIG. 13 shows controls demonstrating Pro104 MAb immunolabelingspecificity.

FIG. 14 shows an epitope map of Pro104 MAbs.

FIG. 15 shows a western blot showing detection of Pro104 protein inmRNA+ cell lines and ovarian tumor tissue (T) but not normal adjacenttissue (N).

FIG. 16 shows that overexpression of Pro104 leads to phosphorylation ofEGF Receptor.

FIG. 17 shows that the Pro104 protein is glycosylated and GPI-Linked.

FIG. 18 shows the surface biotinylation of native Pro104 in cell lines.

FIG. 19 shows retroviral-mediated overexpression of Pro104 protein inRK3E cells.

FIG. 20 shows retroviral-mediated overexpression of Pro104 protein inSKOV3 Cells.

FIG. 21 shows siRNA mediates specific down-regulation of Pro104 proteinin HeLa cells.

FIG. 22 shows siRNA mediates down-regulation of Pro104 protein in CaOV3cells.

FIG. 23 shows Pro104 siRNA specific knockdown of Pro104 mRNA in CaOV3cells.

FIG. 24 shows Pro104 siRNA specific knockdown of Pro104 mRNA in HeLacells.

FIG. 25 shows Pro104 siRNA specific knockdown of Pro104 mRNA in HeLacells, compared to a positive control.

FIG. 26 shows different Pro104 siRNAs inducing specific mRNA knockdownand apoptosis in HeLa cells.

FIG. 27 shows specific knockdown of Pro104 mRNA in HeLa Cells inducingcell death.

FIG. 28 shows specific mRNA knockdown by Pro104 siRNA inducing apoptosisin HeLa cells.

FIG. 29 shows specific knockdown of Pro104 mRNA in CaOV3 Cells inducingapoptosis.

FIG. 30 shows Pro104 siRNA having no effect on apoptosis in cellswithout Pro104 mRNA.

FIG. 31 shows overexpression of Pro104 inducing cell growth in softagar.

FIG. 32 shows Pro104 protease activity is required for cell growth.

FIG. 33 shows knockdown of Pro104 mRNA by siRNA inhibiting growth ofHeLa cells in soft agar.

FIG. 34 shows knockdown of Pro104 mRNA by siRNA inhibiting growth ofHeLa cells in soft agar.

FIG. 35 shows increased growth of human tumor cells over-expressingPro104.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Human “Pro1044” as used herein, refers to a protein of 314 amino acidsthat is expressed on the cell surface as a glycoprotein. The nucleotideand amino acid sequences of Pro104 have been disclosed, e.g.,WO200016805-A1 PA (DIAD-) DIADEXUS Human cancer-specific gene, Pro104;WO9836054-A1 PA (AMRA-) AMRAD Nucleotide sequence of short isoform ofHELA2; and J. D. Hooper et al. Testisin, a new human serine proteaseexpressed by premeiotic testicular germ cells and lost intesticular germcell tumors. Cancer Research 59:3199-3205 (1999)). Pro104 has also beendisclosed in the REFSEQ database as: NM_(—)006799.2 (GI: 21614534) Homosapiens protease, serine, 21 (testisin) (PRSS21), transcript variant 1,mRNA. Refseq gives the following summary of PRSS21 (Pro104):

-   -   This gene encodes a cell-surface anchored serine protease, which        is a member of the trypsin family of serine proteases. It is        predicted to be active on peptide linkages involving the        carboxyl group of lysine or arginine. The protein localizes to        the cytoplasm and the plasma membrane of premeiotic testicular        germ cells and it may be involved in progression of testicular        tumors of germ cell origin. Alternative splicing of this gene        results in three transcript variants encoding three different        isoforms.

The amino acids of Pro104 are presumably located on the cell surface.Pro104 as used herein include allelic variants and conservativesubstitution mutants of the protein which have Pro104 biologicalactivity. Additionally, splice variants may have Pro104 biologicalactivity. The RefSeq accessions for the splice variants referenced aboveinclude: NM_(—)144956.1 (GI: 21614530) Homo sapiens protease, serine, 21(testisin) (PRSS21), transcript variant 2, mRNA; and NM_(—)144957 (GI:21614532) Homo sapiens protease, serine, 21 (testisin) (PRSS21),transcript variant 3, mRNA.

Our findings that Pro104 is apparently associated with the moreaggressive breast, ovarian, pancreatic and lung cancers makes this cellsurface antigen an attractive target for immunotherapy of these andpossibly other tumor types.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g. bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Preferably, the antibody will be purified (1)to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called J chain, and thereforecontains 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(VH) followed by three constant domains (CH) for each of the α, δ and γchains and four CH domains for μ and ε isotypes. Each L chain has at theN-terminus, a variable domain (VL) followed by a constant domain (CL) atits other end.

The VL is aligned with the VH and the CL is aligned with the firstconstant domain of the heavy chain (CHI). Particular amino acid residuesare believed to form an interface between the light chain and heavychain variable domains. The pairing of a VH and VL together forms asingle antigen-binding site. For the structure and properties of thedifferent classes of antibodies, see, e.g., Basic and ClinicalImmunology, 8th edition, Daniel P. Stites, Abba I. Teff and Tristram G.Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 andChapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 1-10-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aP-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the P-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable: region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 5056 (L2) and 89-97 (L3) in the VL, and aroundabout 1-35 (HI), 50-65 (H2) and 95-102 (113) in the VH; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and96-101 (H3) in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a CL and at least heavy chain constant domains, CHI, CH2 andCH3. The constant domains may be native sequence constant domains (e.g.human native sequence constant domains) or amino acid sequence variantthereof. Preferably, the intact antibody has one or more effectorfunctions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (VH), and the firstconstant domain of one heavy chain (CHI). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)2 fragment which roughly corresponds to two disulfide linkedFab fragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CHI domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of 8 Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

The Fc fragment comprises the carboxyterminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., antibody) derived from nature. Suchnative sequence polypeptides can be isolated from nature or can beproduced by recombinant or synthetic means. Thus, a native sequencepolypeptide can have the amino acid sequence of a naturally occurringhuman polypeptide, murine polypeptide, or polypeptide from any othermammalian species.

The term “amino acid sequence variant” refers to a polypeptide that hasamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants of Pro104 willpossess at least about 70% homology with the native sequence Pro104,preferably, at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90% homology, and most preferably atleast 95%. The amino acid sequence variants can possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one which can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, FcFR1.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. Sequence similarity may be measured by any common sequence analysisalgorithm, such as GAP or BESTFIT or other variation Smith-Watermanalignment. See, T. F. Smith and M S. Waterman, J. Mol. Biol. 147:195-197(1981) and W. R. Pearson, Genomics 11:635-650 (1991).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct Biol. 2:593-596 (1992).

As used herein, an anti-Pro104 antibody that “internalizes” is one thatis taken up by (i.e., enters) the cell upon binding to Pro104 on amammalian cell (i.e. cell surface Pro104). The internalizing antibodywill of course include antibody fragments, human or humanized antibodyand antibody conjugate. For therapeutic applications, internalization invivo is contemplated. The number of antibody molecules internalized willbe sufficient or adequate to kill a Pro104-expressing cell, especially aPro104-expressing cancer cell. Depending on the potency of the antibodyor antibody conjugate, in some instances, the uptake of a singleantibody molecule into the cell is sufficient to kill the target cell towhich the antibody binds. For example, certain toxins are highly potentin killing such that internalization of one molecule of the toxinconjugated to the antibody is sufficient to kill the tumor cell.

Whether an anti-Pro104 antibody internalizes upon binding Pro104 on amammalian cell can be determined by various assays including thosedescribed in the experimental examples below. For example, to testinternalization in vivo, the test antibody is labeled and introducedinto an animal known to have Pro104 expressed on the surface of certaincells. The antibody can be radiolabeled or labeled with fluorescent orgold particles, for instance. Animals suitable for this assay include amammal such as a NCR nude mouse that contains a human Pro104-expressingtumor transplant or xenograft, or a mouse into which cells transfectedwith human Pro104 have been introduced, or a transgenic mouse expressingthe human Pro104 transgene. Appropriate controls include animals thatdid not receive the test antibody or that received an unrelatedantibody, and animals that received an antibody to another antigen onthe cells of interest, which antibody is known to be internalized uponbinding to the antigen. The antibody can be administered to the animal,e.g., by intravenous injection. At suitable time intervals, tissuesections of the animal can be prepared using known methods or asdescribed in the experimental examples below, and analyzed by lightmicroscopy or electron microscopy, for internalization as well as thelocation of the internalized antibody in the cell. For internalizationin vitro, the cells can be incubated in tissue culture dishes in thepresence or absence of the relevant antibodies added to the culturemedia and processed for microscopic analysis at desired time points. Thepresence of an internalized, labeled antibody in the cells can bedirectly visualized by microscopy or by autoradiography if radiolabeledantibody is used. Alternatively, in a quantitative biochemical assay, apopulation of cells comprising Pro104-expressing cells are contactedin-vitro or in vivo with a radiolabeled test antibody and the cells (ifcontacted in vivo, cells are then isolated after a suitable amount oftime) are treated with a protease or subjected to an acid wash to removeuninternalized antibody on the cell surface. The cells are ground up andthe amount of protease resistant, radioactive counts per minute (cpm)associated with each batch of cells is measured by passing thehomogenate through a scintillation counter. Based on the known specificactivity of the radiolabeled antibody, the number of antibody moleculesinternalized per cell can be deduced from the scintillation counts ofthe ground-up cells. Cells are “contacted” with antibody in vitropreferably in solution form such as by adding the cells to the cellculture media in the culture dish or flask and mixing the antibody wellwith the media to ensure uniform exposure of the cells to the antibody.Instead of adding to the culture media, the cells can be contacted withthe test antibody in an isotonic solution such as PBS in a test tube forthe desired time period. In vivo, the cells are contacted with antibodyby any suitable method of administering the test antibody such as themethods of administration described below when administered to apatient.

The faster the rate of internalization of the antibody upon binding tothe Pro104-expressing cell in vivo, the faster the desired killing orgrowth inhibitory effect on the target Pro104-expressing cell can beachieved, e.g., by a cytotoxic immunoconjugate. Preferably, the kineticsof internalization of the anti-Pro104 antibodies are such that theyfavor rapid killing of the Pro104-expressing target cell. Therefore, itis desirable that the anti-Pro104 antibody exhibit a rapid rate ofinternalization preferably, within 24 hours from administration of theantibody in vivo, more preferably within about 12 hours, even morepreferably within about 30 minutes to 1 hour, and most preferably,within about 30 minutes. The present invention provides antibodies thatinternalize as fast as about 15 minutes from the time of introducing theanti-Pro104 antibody in vivo. The antibody will preferably beinternalized into the cell within a few hours upon binding to Pro104 onthe cell surface, preferably within 1 hour, even more preferably within15-30 minutes.

To determine if a test antibody can compete for binding to the sameepitope as the epitope bound by the anti-Pro104 antibodies of thepresent invention including the antibodies produced by the hybridomasdeposited with the ATCC, a cross-blocking assay e.g., a competitiveELISA assay can be performed. In an exemplary competitive ELISA assay,Pro104-coated wells of a microtiter plate, or Pro104-coated sepharosebeads, are pre-incubated with or without candidate competing antibodyand then a biotin-labeled anti-Pro104 antibody of the invention isadded. The amount of labeled anti-Pro104 antibody bound to the Pro104antigen in the wells or on the beads is measured using avidin-peroxidaseconjugate and appropriate substrate.

Alternatively, the anti-Pro104 antibody can be labeled, e.g., with aradioactive or fluorescent label or some other detectable and measurablelabel. The amount of labeled anti-Pro104 antibody that binds to theantigen will have an inverse correlation to the ability of the candidatecompeting antibody (test antibody) to compete for binding to the sameepitope on the antigen, i.e., the greater the affinity of the testantibody for the same epitope, the less labeled anti-Pro104 antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as ananti-Pro104 antibody of the invention if the candidate competingantibody can block binding of the anti-Pro104 antibody by at least 20%,preferably by at least 20-50%, even more preferably, by at least 50% ascompared to a control performed in parallel in the absence of thecandidate competing antibody (but may be in the presence of a knownnoncompeting antibody). It will be understood that variations of thisassay can be performed to arrive at the same quantitative value.

An antibody having a “biological characteristic” of a designatedantibody, such as any of the monoclonal antibodies Pro104.C1, Pro104.C4,Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25,Pro104.C27, Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49,Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60,Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9,Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20, Pro104.D21,Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51,Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64,Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111,Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116,Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121,Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125,Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129,Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134,Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139,Pro104.K14, Pro104.K15, Pro104.K16, Pro104K47, Pro104.K71, Pro104.K72,Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78, Pro104.K81, Pro104.K87,Pro104.K88, Pro104.K89, Pro104.K155, Pro104.K156, Pro104.K157,Pro104.K158, Pro104.K159, Pro104.K160, Pro104.K163, Pro104.K164,Pro104.K176, Pro104.K217, Pro104.K226, Pro104.K227, Pro104.K240,Pro104.K274, Pro104.K264, Pro104.K281, Pro104.K358 or Pro104.K362, isone which possesses one or more of the biological characteristics ofthat antibody which distinguish it from other antibodies that bind tothe same antigen, Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17,Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34,Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53,Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60, Pro104.C66, Pro104.C75,Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14,Pro104.D18, Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29,Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56,Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69,Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88, Pro104.D91, Pro104.D94,Pro104.D102, Pro104.D106, Pro104.D111, Pro104.D112, Pro104.D113,Pro104.D114, Pro104.D115, Pro104.D116, Pro104.D117, Pro104.D118,Pro104.D119, Pro104.D120, Pro104.D121, Pro104.D122, Pro104.D123,Pro104.D, Pro104.D124, Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D,Pro104.D128, Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132,Pro104.D133, Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137,Pro104.D138, Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16,Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,Pro104.K358 or Pro04.K362 will bind the same epitope as that bound byPro04.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19,Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37, Pro104.C46,Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55,Pro104.C57, Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43,Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62,Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81,Pro104.D85, Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102,Pro104.D106, Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114,Pro104.D115, Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119,Pro104.D120, Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D,Pro104.D124, Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D,Pro104.D128, Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132,Pro104.D133, Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137,Pro104.D138, Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16,Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,Pro104.K358 or Pro104.K362 (e.g. which competes for binding or blocksbinding of monoclonal antibody Pro104.C1, Pro104.C4, Pro104.C13,Pro104.C17, Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27,Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50,Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60, Pro104.C66,Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9, Pro104.D12,Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26,Pro104.D29, Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55,Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68,Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88, Pro104.D91,Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111, Pro104.D112,Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116, Pro104.D117,Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121, Pro104.D122,Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125, Pro104.D126,Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129, Pro104.D130,Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134, Pro104.D135,Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139, Pro104.K14,Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74,Pro104.K75, Pro104.K76, Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88,Pro104.K89, Pro104.K155, Pro104.K156, Pro104.K157, Pro104.K158,Pro104.K159, Pro104.K160, Pro104.K163, Pro104.K164, Pro104.K176,Pro104.K217, Pro104.K226, Pro104.K227, Pro104.K240, Pro104.K274,Pro104.K264, Pro104.K281, Pro104.K358 or Pro104.K362 to Pro104), be ableto target a Pro104-expressing tumor cell in vivo and will bind to Pro104on a mammalian cell in vivo.

Furthermore, an antibody with the biological characteristic of thePro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19,Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37, Pro104.C46,Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55,Pro104.C57, Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43,Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62,Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81,Pro104.D85, Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102,Pro104.D106, Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114,Pro104.D115, Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119,Pro104.D120, Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D,Pro104.D124, Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D,Pro104.D128, Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132,Pro104.D133, Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137,Pro104.D138, Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16,Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,Pro104.K358 or Pro104.K362 antibody will internalize upon binding toPro104 on a mammalian cell in vivo.

Likewise, an antibody with the biological characteristic of thePro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19,Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37, Pro104.C46,Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55,Pro104.C57, Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4,Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19,Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43,Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62,Pro104.D63, Pro104.D64, Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81,Pro104.D85, Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102,Pro104.D106, Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114,Pro104.D115, Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119,Pro104.D120, Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D,Pro104.D124, Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D,Pro104.D128, Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132,Pro104.D133, Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137,Pro104.D138, Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16,Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,Pro104.K163, Pro104.K164, Pro104.K176, Pro104.K217, Pro104.K226,Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,Pro104.K358 or Pro104.K362 antibody will have the same epitope binding,targeting, internalizing, tumor growth inhibitory and cytotoxicproperties of the antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of a native Pro104 protein disclosedherein. Methods for identifying antagonists of a Pro104 polypeptide maycomprise contacting a Pro104 polypeptide or a cell expressing Pro104 onthe cell surface, with a candidate antagonist antibody and measuring adetectable change in one or more biological activities normallyassociated with the Pro104 polypeptide.

An “antibody that inhibits the growth of tumor cells expressing Pro104”or a “growth inhibitory” antibody is one which binds to and results inmeasurable growth inhibition of cancer cells expressing oroverexpressing Pro104. Preferred growth inhibitory anti-Pro104antibodies inhibit growth of Pro104-expressing tumor cells e.g., breast,ovarian, pancreatic and lung cancer cells) by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g. from about 50% to about 100%) as compared to theappropriate control, the control typically being tumor cells not treatedwith the antibody being tested. Growth inhibition can be measured at anantibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200nM in cell culture, where the growth inhibition is determined 1-10 daysafter exposure of the tumor cells to the antibody. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. The antibody isgrowth inhibitory in vivo if administration of the anti-Pro104 antibodyat about 1 pg/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3 monthsfrom the first administration of the antibody, preferably within about 5to 30 days.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses Pro104. Preferably the cell is atumor cell, e.g. an breast, ovarian, pancreatic and lung cell. Variousmethods are available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody which induces apoptosis is one which results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcells in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al. PNAS (ISA) 95:652-656(1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRI, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based-inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126.330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer, of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996) may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, multiple myeloma and B-cell lymphoma, brain, as well as headand neck cancer, and associated metastases.

A “Pro104-expressing cell” is a cell which expresses endogenous ortransfected Pro104 on the cell surface. A “Pro104-expressing cancer” isa cancer comprising cells that have Pro104 protein present on the cellsurface. A “Pro104-expressing cancer” produces sufficient levels ofPro104 on the surface of cells thereof, such that an anti-Pro104antibody can bind thereto and have a therapeutic effect with respect tothe cancer. A cancer which “overexpresses” Pro104 is one which hassignificantly higher levels of Pro104 at the cell surface thereof,compared to a noncancerous cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. Pro104 overexpression may be determined ina diagnostic or prognostic assay by evaluating increased levels of thePro104 protein present on the surface of a cell (e.g. via animmunohistochemistry assay; FACS analysis). Alternatively, oradditionally, one may measure levels of Pro104-encoding nucleic acid ormRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; seeWO98/45479 published October, 1998), Southern blotting, Northernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). One may also study Pro104 overexpressionby measuring shed antigen in a biological fluid such as serum, e.g.,using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No.5,401,638 issued Mar. 28, 1995; and Sias et al. J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays, various in vivo assays areavailable to the skilled practitioner. For example, one may expose cellswithin the body of the patient to an antibody which is optionallylabeled with a detectable label, e.g. a radioactive isotope, and bindingof the antibody to cells in the patient can be evaluated, e.g. byexternal scanning for radioactivity or by analyzing a biopsy taken froma patient previously exposed to the antibody. A Pro104-expressing cancerincludes ovarian, pancreatic, lung or breast cancer.

A “mammal” for purposes of treating a cancer or alleviating the symptomsof cancer, refers to any mammal, including-humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal ishuman.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for a Pro104-expressing cancer if, after receiving atherapeutic amount of an anti-Pro104 antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer, reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe anti-Pro104 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of these signsor symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR).

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See preceding definition of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed.

Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, e.g., gelonin,ricin, saporin, and the various antitumor or anticancer agents disclosedbelow. Other cytotoxic agents are described below. A tumoricidal agentcauses destruction of tumor cells.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially aPro104-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of Pro104-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce GI arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semi synthetic analogue of paclitaxel (FAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Label” as used herein refers to a detectable compound or compositionwhich is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g. radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term “epitope tagged” used herein refers to a chimeric polypeptidecomprising an anti-Pro104 antibody polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the Ig polypeptide to whichit is fused. The tag polypeptide is also preferably fairly unique sothat the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated nucleic acid molecule” is a nucleic acid molecule, e.g., anRNA, DNA, or a mixed polymer, which is substantially separated fromother genome DNA sequences as well as proteins or complexes such asribosomes and polymerases, which naturally accompany a native sequence.The term embraces a nucleic acid molecule which has been removed fromits naturally occurring environment, and includes recombinant or clonedDNA isolates and chemically synthesized analogues or analoguesbiologically synthesized by heterologous systems. A substantially purenucleic acid molecule includes isolated forms of the nucleic acidmolecule.

“Vector” includes shuttle and expression vectors and includes, e.g., aplasmid, cosmid, or phagemid. Typically, a plasmid construct will alsoinclude an origin of replication (e.g., the ColE1 origin of replication)and a selectable marker (e.g., ampicillin or tetracycline resistance),for replication and selection, respectively, of the plasmids inbacteria. An “expression vector” refers to a vector that contains thenecessary control sequences or regulatory elements for expression of theantibodies including antibody fragment of the invention, in prokaryotic,e.g., bacterial, or eukaryotic cells. Suitable vectors are disclosedbelow.

The cell that produces an anti-Pro104 antibody of the invention willinclude the parent hybridoma cell e.g., the hybridomas that aredeposited with the ATCC, as well as bacterial and eukaryotic host cellsinto which nucleic acid encoding the antibodies have been introduced.Suitable host cells are disclosed below.

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The correspondingprocess in plants is commonly referred to as post transcriptional genesilencing or RNA silencing and is also referred to as quelling in fungi.The process of post transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologous singlestranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA mediated activation ofprotein kinase PKR and 2′,5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from dicer activity are typically about21-23 nucleotides in length and comprise about 19 base pair duplexes.Dicer has also been implicated in the excision of 21 and 22 nucleotidesmall temporal RNAs (stRNA) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

Short interfering RNA mediated RNAi has been studied in a variety ofsystems. Fire et al., 1998, Nature, 391, 806, were the first to observeRNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70,describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,Nature, 404, 293, describe RNAi in Drosophila cells transfected withdsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two nucleotide3′-overhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAIactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashiret al., 2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of a siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-overhanging segments of a21-mer siRNA duplex having 2 nucleotide 3′ overhangs withdeoxyribonucleotides does not have an adverse effect on RNAI activity.Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAI activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 both suggest that siRNA“may include modifications to either the phosphate-sugar back bone orthe nucleoside to include at least one of a nitrogen or sulfurheteroatom”, however neither application teaches to what extent thesemodifications are tolerated in siRNA molecules nor provide any examplesof such modified siRNA. Kreutzer and Limmer, Canadian Patent ApplicationNo. 2,359,180, also describe certain chemical modifications for use indsRNA constructs in order to counteract activation of doublestranded-RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer and Limmer similarly fail to show towhat extent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that “RNAs with two (phosphorothioate) modified bases also hadsubstantial decreases in effectiveness as RNAi triggers (data notshown); (phosphorothioate) modification of more than two residuesgreatly destabilized the RNAs in vitro and we were not able to assayinterference activities.” Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and observed that substituting deoxynucleotides forribonucleotides “produced a substantial decrease in interferenceactivity”, especially in the case of Uridine to Thymidine and/orCytidine to deoxy-Cytidine substitutions. Id. In addition, the authorstested certain base modifications, including substituting 4-thiouracil,5-bromouracil, 5-iodouracil, 3-(aminoallyl)uracil for uracil, andinosine for guanosine in sense and antisense strands of the siRNA, andfound that whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine “produced a substantial decrease in interferenceactivity” when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resultedin substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describesspecific methods for attenuating gene expression using endogenouslyderived dsRNA. Tuschl et al., International PCT Publication No. WO01/75164, describes a Drosophila in vitro RNAi system and the use ofspecific siRNA molecules for certain functional genomic and certaintherapeutic applications; although Tuschl, 2001, Chem. Biochem., 2,239-245, doubts that RNAi can be used to cure genetic diseases or viralinfection due “to the danger of activating interferon response”. L1 etal., International PCT Publication No. WO 00/44914, describes the use ofspecific dsRNAs for use in attenuating the expression of certain targetgenes. Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expression ofparticular genes in mammalian cells using certain dsRNA molecules. Fireet al., International PCT Publication No. WO 99/32619, describesparticular methods for introducing certain dsRNA molecules into cellsfor use in inhibiting gene expression. Plaetinck et al., InternationalPCT Publication No. WO 00/01846, describes certain methods foridentifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describes theidentification of specific genes involved in dsRNA mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Driscoll et al.,International PCT Publication No. WO 01/49844, describes specific DNAconstructs for use in facilitating gene silencing in targeted organisms.Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specificchemically modified siRNA constructs targeting the unc-22 gene of C.elegans. Tuschl et al., International PCT Publication No. WO 02/44321,describe certain synthetic siRNA constructs.

Compositions and Methods of the Invention

The invention provides anti-Pro104 antibodies. Preferably, theanti-Pro104 antibodies internalize upon binding to cell surface Pro104on a mammalian cell. The anti-Pro104 antibodies may also destroy or leadto the destruction of tumor cells bearing Pro104.

It was not apparent that Pro104 was internalization-competent. Inaddition the ability of an antibody to internalize depends on severalfactors including the affinity, avidity, and isotype of the antibody,and the epitope that it binds. We have demonstrated herein that the cellsurface Pro104 is internalization competent upon binding by theanti-Pro104 antibodies of the invention. Additionally, it wasdemonstrated that the anti-Pro104 antibodies of the present inventioncan specifically target Pro104-expressing tumor cells in vivo andinhibit or kill these cells. These in vivo tumor targeting,internalization and growth inhibitory properties of the anti-Pro104antibodies make these antibodies very suitable for therapeutic uses,e.g., in the treatment of various cancers including breast, ovarian,pancreatic and lung cancer. Internalization of the anti-Pro104 antibodyis preferred, e.g., if the antibody or antibody conjugate has anintracellular site of action and if the cytotoxic agent conjugated tothe antibody does not readily cross the plasma membrane (e.g., the toxincalicheamicin). Internalization is not necessary if the antibodies orthe agent conjugated to the antibodies do not have intracellular sitesof action, e.g., if the antibody can kill the tumor cell by ADCC or someother mechanism.

The anti-Pro104 antibodies of the invention also have variousnon-therapeutic applications. The anti-Pro104 antibodies of the presentinvention can be useful for diagnosis and staging of Pro104-expressingcancers (e.g., in radioimaging). They may be used alone or incombination with other ovarian cancer markers, including, but notlimited to, CA125, HE4 and mesothelin. The antibodies are also usefulfor purification or immunoprecipitation of Pro104 from cells, fordetection and quantitation of Pro104 in vitro, e.g. in an ELISA or aWestern blot, to kill and eliminate Pro104-expressing cells from apopulation of mixed cells as a step in the purification of other cells.The internalizing anti-Pro104 antibodies of the invention can be in thedifferent forms encompassed by the definition of “antibody” herein.Thus, the antibodies include full length or intact antibody, antibodyfragments, native sequence antibody or amino acid variants, humanized,chimeric or fusion antibodies, immunoconjugates, and functionalfragments thereof. In fusion antibodies, an antibody sequence is fusedto a heterologous polypeptide sequence. The antibodies can be modifiedin the Fc region to provide desired effector functions. As discussed inmore detail in the sections below, with the appropriate Fc regions, thenaked antibody bound on the cell surface can induce cytotoxicity, e.g.,via antibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate or reduceeffector function, so as to minimize side effects or therapeuticcomplications, certain other Fc regions may be used.

The antibody may compete for binding, or binds substantially to, thesame epitope bound by the antibodies of the invention. Antibodies havingthe biological characteristics of the present anti-Pro104 antibodies ofthe invention are also contemplated, e.g., an anti-Pro104 antibody whichhas the biological characteristics of a monoclonal antibody produced bythe hybridomas accorded ATCC accession numbers PTA-5277, 6076, 6077 and6078, specifically including the in vivo tumor targeting,internalization and any cell proliferation inhibition or cytotoxiccharacteristics. Specifically provided are anti-Pro104 antibodies thatbind to an epitope present in amino acids 1-10, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130,130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210,210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290,290-300, 300-310, 310-314 of human Pro104.

Methods of producing the above antibodies are described in detail below.

The present anti-Pro104 antibodies are useful for treating aPro104-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such cancers include ovarian and pancreatic cancer,cancer of the urinary tract, prostate cancer, breast cancer, coloncancer, and lung cancer. Such a cancer includes more specifically,ovarian serous adenocarcinoma, breast infiltrating ductal carcinoma,prostate adenocarcinoma, renal cell carcinomas, colorectaladenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas,and pleural mesothelioma. The breast cancer may be HER-2 negative orpositive breast cancer. The cancers encompass metastatic cancers of anyof the preceding, e.g., breast, ovarian, pancreatic and lung cancermetastases. The antibody is able to bind to at least a portion of thecancer cells that express Pro104 in the mammal and preferably is onethat does not induce or that minimizes HAMA response. Preferably, theantibody is effective to destroy or kill Pro104-expressing tumor cellsor inhibit the growth of such tumor cells, in vitro or in vivo, uponbinding to Pro104 on the cell. Such an antibody includes a nakedanti-Pro104 antibody (not conjugated to any agent). Naked anti-Pro104antibodies having tumor growth inhibition properties in vivo include theantibodies described in the Experimental Examples below. Nakedantibodies that have cytotoxic or cell growth inhibition properties canbe further conjugated with a cytotoxic agent to render them even morepotent in tumor cell destruction. Cytotoxic properties can be conferredto an anti-Pro104 antibody by, e.g., conjugating the antibody with acytotoxic agent, to form an immunoconjugate as described below. Thecytotoxic agent or a growth inhibitory agent is preferably a smallmolecule. Toxins such as maytansin, maytansinoids, saporin, gelonin,ricin or calicheamicin and analogs or derivatives thereof, arepreferable.

The invention provides a composition comprising an anti-Pro104 antibodyof the invention, and a carrier. For the purposes of treating cancer,compositions can be administered to the patient in need of suchtreatment, wherein the composition can comprise one or more anti-Pro104antibodies present as an immunoconjugate or as the naked antibody.Further, the compositions can comprise these antibodies in combinationwith other therapeutic agents such as cytotoxic or growth inhibitoryagents, including chemotherapeutic agents. The invention also providesformulations comprising an anti-Pro104 antibody of the invention, and acarrier. The formulation may be a therapeutic formulation comprising apharmaceutically acceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding theinternalizing anti-Pro104 antibodies. Nucleic acids encoding both the Hand L chains and especially the hypervariable region residues, chainswhich encode the native sequence antibody as well as variants,modifications and humanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating aPro104-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an internalizing anti-Pro104 antibody to the mammal. Theantibody therapeutic compositions can be administered short term (acute)or chronic, or intermittent as directed by physician. Also provided aremethods of inhibiting the growth of, and killing a Pro104 expressingcell. Finally, the invention also provides kits and articles ofmanufacture comprising at least one antibody of this invention,preferably at least one anti-Pro104 antibody of this invention thatbinds to Pro104 on a mammalian cell in vivo or at least oneinternalizing anti-Pro104 antibody of this invention. Kits containinganti-Pro104 antibodies find use in detecting Pro104 expression, or intherapeutic or diagnostic assays, e.g., for Pro104 cell killing assaysor for purification and/or immunoprecipitation of Pro104 from cells. Forexample, for isolation and purification of Pro104, the kit can containan anti-Pro104 antibody coupled to a solid support, e.g., a tissueculture plate or beads (e.g., sepharose beads). Kits can be providedwhich contain antibodies for detection and quantitation of Pro104 invitro, e.g. in an ELISA or a Western blot. Such antibody useful fordetection may be provided with a label such as a fluorescent orradiolabel.

Production of Anti-Pro104 Antibodies

The following describes exemplary techniques for the production of theantibodies useful in the present invention. Some of these techniques aredescribed further in Example 1. The Pro104 antigen to be used forproduction of antibodies may be, e.g., the full length polypeptide or aportion thereof, including a soluble form of Pro104 lacking the membranespanning sequence, or synthetic peptides to selected portions of theprotein.

Alternatively, cells expressing Pro104 at their cell surface (e.g. CHOor NIH-3T3 cells transformed to overexpress Pro104; ovarian, pancreatic,lung, breast or other Pro104-expressing tumor cell line), or membranesprepared from such cells can be used to generate antibodies. Thenucleotide and amino acid sequences of human and murine Pro104 areavailable as provided above. Pro104 can be produced recombinantly in andisolated from, prokaryotic cells, e.g., bacterial cells, or eukaryoticcells using standard recombinant DNA methodology. Pro104 can beexpressed as a tagged (e.g., epitope tag) or other fusion protein tofacilitate its isolation as well as its identification in variousassays.

Antibodies or binding proteins that bind to various tags and fusionsequences are available as elaborated below. Other forms of Pro104useful for generating antibodies will be apparent to those skilled inthe art.

Tags

Various tag polypeptides and their respective antibodies are well knownin the art Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165(1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990)). The FLAG-peptide (Hopp et al., BioTechnology, 6:1204-1210(1988)) is recognized by an anti-FLAG M2 monoclonal antibody (EastmanKodak Co., New Haven, Conn.). Purification of a protein containing theFLAG peptide can be performed by immunoaffinity chromatography using anaffinity matrix comprising the anti-FLAG M2 monoclonal antibodycovalently attached to agarose (Eastman Kodak Co., New Haven, Conn.).Other tag polypeptides include the KT3 epitope peptide [Martin et al.,Science, 255:192-194 (1992)]; an α-tubulin epitope peptide (Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)); and the T7 gene proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)).

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals, preferablynon-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip)injections of the relevant antigen and an adjuvant. It may be useful toconjugate the relevant antigen (especially when synthetic peptides areused) to a protein that is immunogenic in the species to be immunized.For example, the antigen can be conjugated to keyhole limpet hemocyanin(KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, usinga bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkylgroups. Conjugates also can be made in recombinant cell culture asprotein fusions.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 5-100 pg of the protein or conjugate(for rabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later, the animals are boosted with ⅕ to 1/10the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later, the animals arebled and the serum is assayed for antibody titer. Animals are boosteduntil the titer plateaus. Also, aggregating agents such as alum aresuitably used to enhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as a hamster, isimmunized as described above to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. After immunization, lymphocytes are isolated andthen fused with a “fusion partner”, e.g., a myeloma cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies. Principles and Practice, pp 103(Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, fusion partner,e.g., the parental myeloma cells. For example, if the parental myelomacells lack the enzyme hypoxanthine guanine phosphoribosyl transferase(HGPRT or HPRT), the selective culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-IImouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Rockville, Md. USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980). Once hybridoma cells that produce antibodiesof the desired specificity, affinity, and/or activity are identified,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, pp 103 (Academic Press, 1986)). Suitable culture media forthis purpose include, for example, D-MEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal e.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transformed or transfected intoprokaryotic or eukaryotic host cells such as, e.g., E coli cells, simianCOS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, that donot otherwise produce antibody protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) andPhickthun, Immunol. Revs., 130:151-188(1992).

Further, the monoclonal antibodies or antibody fragments can be isolatedfrom antibody phage libraries generated using the techniques describedin McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991) describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (CH and CL) sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenonimmunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is nonhuman. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.,151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art.

Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

Various forms of a humanized anti-Pro104 antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fallrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and Alternatively,phage display technology (McCafferty et al., Nature 348:552-553 (1990))can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B-cell. Phage display can be performed in a variety offormats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571 (1993). Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Marks et al., J. Mol. Biol.222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See,also, U.S. Pat. Nos. 5,565,332 and 5,573,905. As discussed above, humanantibodies may also be generated by in vitro activated B cells (see U.S.Pat. Nos. 5,567,610 and 5,229,275).

Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors. Various techniques have been developed for the productionof antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117 (1992); andBrennan et al., Science, 229:81 (1985)). However, these fragments cannow be produced directly by recombinant host cells. Fab, Fv and ScFvantibody fragments can all be expressed in and secreted from E coli,thus allowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab)₂ fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab)₂ fragment with increased in vivohalf-life comprising a salvage-receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice may also be a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat.No. 5,587,458. Fv and sFv are the only species with intact combiningsites that are devoid of constant regions; thus, they are suitable forreduced nonspecific binding during in vivo use. sFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the Pro104 protein. Other suchantibodies may combine a Pro104 binding site with a binding site foranother protein. Alternatively, an anti-Pro104.Arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aTcell receptor molecule (e.g. C133), or Fc receptors for IgG (FcγcR),such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focusand localize cellular defense mechanisms to the Pro104-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express Pro104. These antibodies possess a Pro104-bindingarm and an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab)2 bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (CM) containing the site necessaryfor light chain bonding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. This providesfor greater flexibility in adjusting the mutual proportions of the threepolypeptide fragments in embodiments when unequal ratios of the threepolypeptide chains used in the construction provide the optimum yield ofthe desired bispecific antibody. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chain combination.

Preferably, the bispecific antibodies in this approach are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210(1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers.

The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a VH connected to a VL by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1(X1 n-VD2-X2)n-Fc,wherein VDI is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, XI and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fcregion chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the anti-Pro104 antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of the anti-Pro104 antibody areprepared by introducing appropriate nucleotide changes into theanti-Pro104 antibody nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into, and/or substitutions of, residues within the amino acidsequences of the anti-Pro104 antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe anti-Pro104 antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-Pro104 anti-body that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis” as described by Cunningham andWells in Science, 244:1081-1085 (1989). Here, a residue or group oftarget residues within the anti-Pro104 antibody are identified (e.g.,charged residues such as arg, asp, his, lys, and glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with Pro104antigen.

Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at a target codon or region and theexpressed anti-Pro104 antibody variants are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-Pro104 antibody with an N-terminal methionyl residue or theantibody fused to a cytotoxic polypeptide. Other insertional variants ofthe anti-Pro104 antibody molecule include the fusion to the N- orC-terminus of the anti-Pro104 antibody to an enzyme (e.g. for ADEPT) ora fusion to a polypeptide which increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-Pro104antibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but FR alterations are also contemplated. Conservativesubstitutions are shown in Table 1 under the heading of “preferredsubstitutions”. If such substitutions result in a change in biologicalactivity, then more substantial changes, denominated “exemplarysubstitutions” in Table 1, or as further described below in reference toamino acid classes, may be introduced and the products screened for adesired characteristic. TABLE I Amino Acid Substitutions PreferredOriginal Exemplary Substitutions Substitutions Ala (A) val; leu; ile ValArg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D)glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; glnasp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val;met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ile Lys (K)arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala; leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutralhydrophilic: cys, ser, thr, (3) acidic: asp, glu; (4) basic: asn, gin,his, lys, arg; (5) residues that influence-chain orientation: gly, pro;and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the anti-Pro104 antibody also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibody variants thus generated are displayed in a monovalent fashionfrom filamentous phage particles as fusions to the gene III product ofM13 packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human Pro104. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for 6-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-Pro104 antibody are prepared by a variety of methods known in theart These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared nucleic acid molecule encoding a variant or a non-variantversion of the anti-Pro104 antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.

See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of the antibody.

Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfarther select antibodies with certain biological characteristics, asdesired.

The growth inhibitory effects of an anti-Pro104 antibody of theinvention may be assessed by methods known in the art e.g., using cellswhich express Pro104 either endogenously or following transfection withthe Pro104 gene. For example, the tumor cell lines andPro104-transfected cells provided in Example 1 below may be treated withan anti-Pro104 monoclonal antibody of the invention at variousconcentrations for a few days (e.g., 2-7) days and stained with crystalviolet or MTT or analyzed by some other calorimetric assay. Anothermethod of measuring proliferation would be by comparing ³H-thymidineuptake by the cells treated in the presence or absence an anti-Pro104antibody of the invention. After antibody treatment, the cells areharvested and the amount of radioactivity incorporated into the DNAquantitated in a scintillation counter. Appropriated positive controlsinclude treatment of a selected cell line with a growth inhibitoryantibody known to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. Preferably, thetumor cell is one that over-expresses Pro104. Preferably, theanti-Pro104 antibody will inhibit cell proliferation of aPro104-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, more preferably, by about 30-100%,and even more preferably by about 50-100% or 70-100%, at an antibodyconcentration of about 0.5 to 30 μg/ml. Growth inhibition can bemeasured at an antibody concentration of about 0.5 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.The antibody is growth inhibitory in vivo if administration of theanti-Pro104 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

To select for antibodies which induce cell death, loss of membraneintegrity as indicated by, e.g., propidium iodide (P), trypan blue or7AAD uptake may be assessed relative to a control. A PI uptake assay canbe performed in the absence of complement and immune effector cells.Pro104-expressing tumor cells are incubated with medium alone or mediumcontaining of the appropriate monoclonal antibody at e.g., about 10μg/ml. The cells are incubated for a 3 day time period. Following eachtreatment, cells are washed and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake may beselected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on Pro104 bound by anantibody of interest, e.g., the Pro104 antibodies of this invention, aroutine cross-blocking assay such as that describe in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if atest antibody binds the same site or epitope as an anti-Pro104 antibodyof the invention. Alternatively, or additionally, epitope mapping can beperformed by methods known in the art. For example, the antibodysequence can be mutagenized such as by alanine scanning, to identifycontact residues. The mutant antibody is initially tested for bindingwith polyclonal antibody to ensure proper folding. In a differentmethod, peptides corresponding to different regions of Pro104 can beused in competition assays with the test antibodies or with a testantibody and an antibody with a characterized or known epitope.

For example, a method to screen for antibodies that bind to an epitopewhich is bound by an antibody this invention may comprise combining aPro104-containing sample with a test antibody and an antibody of thisinvention to form a mixture, the level of Pro104 antibody bound toPro104 in the mixture is then determined and compared to the level ofPro104 antibody bound in the mixture to a control mixture, wherein thelevel of Pro104 antibody binding to Pro104 in the mixture as compared tothe control is indicative of the test antibody's binding to an epitopethat is bound by the anti-Pro104 antibody of this invention. The levelof Pro104 antibody bound to Pro104 is determined by ELISA. The controlmay be a positive or negative control or both. For example, the controlmay be a mixture of Pro104, Pro104 antibody of this invention and anantibody known to bind the epitope bound by the Pro104 antibody of thisinvention. The anti-Pro104 antibody labeled with a label such as thosedisclosed herein. The Pro104 may be bound to a solid support, e.g., atissue culture plate or to beads, e.g., sepharose beads.

Immunoconjugates

The invention also pertains to therapy with immunoconjugates comprisingan antibody conjugated to an anti-cancer agent such as a cytotoxic agentor a growth inhibitory agent.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin,maytansinoids, a trichothene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

Preferably, an anti-Pro104 antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the cast Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their-therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al. CancerResearch 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via A disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the BER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10 5 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-Pro104 Antibody-Maytansinoid Conjugates (Immunoconjugates)

Anti-Pro104 antibody-maytansinoid conjugates are prepared by chemicallylinking an anti-Pro104 antibody to a maytansinoid molecule withoutsignificantly diminishing the biological activity of either the antibodyor the maytansinoid molecule. An average of 3-4 maytansinoid moleculesconjugated per antibody molecule has shown efficacy in enhancingcytotoxicity of target cells without negatively affecting the functionor solubility of the antibody, although even one molecule oftoxin/antibody would be expected to enhance cytotoxicity over the use ofnaked antibody. Maytansinoids are well known in the art and can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove. Preferred maytansinoids are maytansinol and maytansinolanalogues modified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al. Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred. Conjugates of the antibody and maytansinoid maybe made using a variety of bifunctional protein coupling agents such asN-succinimidyl (2-pyridyidithio) propionate (SPDP),succidyl-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane(FI), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as his(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl (2-pyridyldithio) propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) and N-succinimidyl(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. Preferably, the linkage is formedat the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-Pro104 antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁ ^(I), (Himnan etal. Cancer Research 53: 3336 (1993), Lode et al. Cancer Research 5 8:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-Pro104antibodies of the invention include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296). Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain, 15nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, WO 93/21232 published Oct. 28, 1993. The presentinvention further contemplates an immunoconjugate formed between anantibody and a compound with nucleolytic activity (e.g. a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-Pro104 antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³²,and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example Tc^(99M) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc^(99M), I¹²³, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such as N-succinimidyl(2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (T), bifunctional derivativesof imidoesters (such as dimethyl adipimidate HCL), active esters (suchas disuccinimidyl suberate), aldehydes (such as glutaraldehyde),bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al. Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be uses.

Alternatively, a fusion protein comprising the anti-Pro104 antibody andcytotoxic agent may be made, e.g. by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

In addition, the antibody may be conjugated to a “receptor” (suchstreptavidin) for utilization in tumor pre-targeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide).

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosinie deaminase useful for convertingnon-toxic fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asO-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; P-lactamase useful for converting drugsderivatized with P-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population. The enzymes of this invention can becovalently bound to the anti-Pro104 antibodies by techniques well knownin the art such as the use of the heterobifunctional crosslinkingreagents discussed above.

Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 31.2: 604-608 (1984).

Other Antibody Modifications

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-Pro104 antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acid molecule encoding thehumanized anti-Pro104 antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody. For recombinant production of the antibody, the nucleic acidmolecule encoding it is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or inserted into a vectorin operable linkage with a promoter for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to nucleic acid molecules encoding the heavy andlight chains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Signal Sequence Component

The anti-Pro104 antibody of this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native anti-Pro104 antibody signal sequence,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g., theyeast invertase leader, oc factor leader (including Saccharomyces andKluyveromyces cc-factor leaders), or acid phosphatase leader, the Calbicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor region is ligated in reading frameto DNA encoding the anti-Pro104 antibody.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theanti-Pro104 antibody nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -11, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc. For example, cellstransformed with the DHFR selection gene are first identified byculturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DBFR) transformed or co-transformed with DNA sequencesencoding anti-Pro104 antibody, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4 Jones, Genetics, 85:12 (1977). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDI canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theanti-Pro104 antibody nucleic acid. Promoters suitable for use withprokaryotic hosts include the phoA promoter, P-lactamase and lactosepromoter systems, alkaline phosphatase promoter, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the anti-Pro104 antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors. Examples of suitable promoter sequences for use withyeast hosts include the promoters for 3-phosphoglycerate kinase or otherglycolytic enzymes, such as enolase, glyceraldehyde phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Anti-Pro104 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human P-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding the anti-Pro104 antibody of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theanti-Pro104 antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-Pro104 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO 94/11026 and the expression vectordisclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation region (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-Pro104antibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-Pro104antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,Arabidopsis and tobacco can also be utilized as hosts. Cloning andexpression vectors useful in the production of proteins in plant cellculture are known to those of skill in the art. See e.g. Hiatt et al.,Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794,Artsaenko et al. (1995) The Plant J 8: 745-750, and Fecker et al. (1996)Plant Mol Biol 32: 979-986.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; S4 cells; and a human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning Vectors for anti-Pro104 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

Culturing Host Cells

The host cells used to produce the anti-Pro104 antibody of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's FIO (Sigma), Minimal Essential Medium (MEM)(Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM)(Sigma)are suitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Anti-Pro104 Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of Ecoli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30min. Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Anicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SIDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Pharmaceutical formulations of the antibodies used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate, Tris,phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanoli and mcresol); low molecular weight(less, than about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyllolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theantibody preferably comprises the antibody at a concentration of between5-200 mg/ml, preferably between 10-100 mg/ml.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, in addition to the anti-Pro104 antibody which internalizes,it may be desirable to include in the one formulation, an additionalantibody, e.g. a second anti-Pro104 antibody which binds a differentepitope on Pro104, or an antibody to some other target such as a growthfactor that affects the growth of the particular cancer. Alternatively,or additionally, the composition may further comprise a chemotherapeuticagent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonalagent, and/or cardioprotectant. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively,in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules) or inmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutarnate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−) hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Methods and Treatment Using Anti-Pro104 Antibodies

According to the present invention, the anti-Pro104 antibody thatinternalizes upon binding Pro104 on a cell surface is used to treat asubject in need thereof having a cancer characterized byPro104-expressing cancer cells, in particular, ovarian, pancreatic, lungor breast cancer, such as ovarian serous adenocarcinoma or breastinfiltrating ductal carcinoma cancer, and associated metastases.

The cancer will generally comprise Pro104-expressing cells, such thatthe anti-Pro104 antibody is able to bind thereto. While the cancer maybe characterized by overexpression of the Pro104 molecule, the presentapplication further provides a method for treating cancer which is notconsidered to be a Pro104-overexpressing cancer.

This invention also relates to methods for detecting cells whichoverexpress Pro104 and to diagnostic kits useful in detecting cellsexpressing Pro104 or in detecting Pro104 in serum from a patient. Themethods may comprise combining a cell-containing test sample with anantibody of this invention, assaying the test sample for antibodybinding to cells in the test sample and comparing the level of antibodybinding in the test sample to the level of antibody binding in a controlsample of cells. A suitable control is, e.g., a sample of normal cellsof the same type as the test sample or a cell sample known to be free ofPro104 overexpressing cells. A level of Pro104 binding higher than thatof such a control sample would be indicative of the test samplecontaining cells that overexpress Pro104. Alternatively the control maybe a sample of cells known to contain cells that overexpress Pro104. Insuch a case, a level of Pro104 antibody binding in the test sample thatis similar to, or in excess of, that of the control sample would beindicative of the test sample containing cells that overexpress Pro104.

Pro104 overexpression may be detected with a various diagnostic assays.For example, over expression of Pro104 may be assayed byimmunohistochemistry (IHC). Parrafin embedded tissue sections from atumor biopsy may be subjected to the IHC assay and accorded a Pro104protein staining intensity criteria as follows.

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.

Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for Pro104 expression may becharacterized as not overexpressing Pro104, whereas those tumors with 2+or 3+ scores may be characterized as overexpressing Pro104.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Arizona) or PATHVISION™ (VySiS, Illinois) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of Pro104 overexpression in the tumor. Pro104 overexpression oramplification may be evaluated using an in vivo diagnostic assay, e.g.by administering a molecule (such as an antibody of this invention)which binds Pro104 and which is labeled with a detectable label (e.g. aradioactive isotope or a fluorescent label) and externally scanning thepatient for localization of the label.

A sample suspected of containing cells expressing or overexpressingPro104 is combined with the antibodies of this invention underconditions suitable for the specific binding of the antibodies toPro104. Binding and/or internalizing the Pro104 antibodies of thisinvention is indicative of the cells expressing Pro104. The level ofbinding may be determined and compared to a suitable control, wherein anelevated level of bound Pro104 as compared to the control is indicativeof Pro104 overexpression. The sample suspected of containing cellsoverexpressing Pro104 may be a cancer cell sample, particularly a sampleof an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breastcancer, e.g., a breast infiltrating ductal carcinoma. A serum samplefrom a subject may also be assayed for levels of Pro104 by combining aserum sample from a subject with a Pro104 antibody of this invention,determining the level of Pro104 bound to the antibody and comparing thelevel to a control, wherein an elevated level of Pro104 in the serum ofthe patient as compared to a control is indicative of overexpression ofPro104 by cells in the patient. The subject may have a cancer such ase.g., an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breastcancer, e.g., a breast infiltrating ductal carcinoma.

Currently, depending on the stage of the cancer, ovarian, pancreatic,lung or breast cancer treatment involves one or a combination of thefollowing therapies: surgery to remove the cancerous tissue, radiationtherapy, androgen deprivation (e.g., hormonal therapy), andchemotherapy. Anti-Pro104 antibody therapy may be especially desirablein elderly patients who do not tolerate the toxicity and side effects ofchemotherapy well, in metastatic disease where radiation therapy haslimited usefulness, and for the management of prostatic carcinoma thatis resistant to androgen deprivation treatment. The tumor targeting andinternalizing anti-Pro104 antibodies of the invention are useful toalleviate Pro104-expressing cancers, e.g., ovarian, pancreatic, lung orbreast cancers upon initial diagnosis of the disease or during relapse.For therapeutic applications, the anti-Pro104 antibody can be usedalone, or in combination therapy with, e.g., hormones, antiangiogens, orradiolabelled compounds, or with surgery, cryotherapy, and/orradiotherapy, notably for ovarian, pancreatic, lung or breast cancers,also particularly where shed cells cannot be reached. Anti-Pro104antibody treatment can be administered in conjunction with other formsof conventional therapy, either consecutively with, pre- orpost-conventional therapy, Chemotherapeutic drugs such as Taxotere®(docetaxel), Taxol® (paclitaxel), estramustine and mitoxantrone are usedin treating metastatic and hormone refractory ovarian, pancreatic, lungor breast cancer, in particular, in good risk patients. In the presentmethod of the invention for treating or alleviating cancer, inparticular, androgen independent and/or metastatic ovarian, pancreatic,lung or breast cancer, the cancer patient can be administeredanti-Pro104 antibody in conjunction with treatment with the one or moreof the preceding chemotherapeutic agents. In particular, combinationtherapy with palictaxel and modified derivatives (see, e.g., EP0600517)is contemplated. The anti-Pro104 antibody will be administered with atherapeutically effective dose of the chemotherapeutic agent. Theanti-Pro104 antibody may also be administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

Particularly, an immunoconjugate comprising the anti-Pro104 antibodyconjugated with a cytotoxic agent may be administered to the patientPreferably, the immunoconjugate bound to the Pro104 protein isinternalized by the cell, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the cancer cell to which it binds.Preferably, the cytotoxic agent targets or interferes with the nucleicacid in the cancer cell. Examples of such cytotoxic agents are describedabove and include maytansin, maytansinoids, saporin, gelonin, ricin,calicheamicin, ribonucleases and DNA endonucleases.

The anti-Pro104 antibodies or immunoconjugates are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies or immunoconjugates may beinjected directly into the tumor mass. Intravenous or subcutaneousadministration of the antibody is preferred. Other therapeutic regimensmay be combined with the administration of the anti-Pro104 antibody.

The combined administration includes co-administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities. Preferably such combined therapy results in asynergistic therapeutic effect.

It may also be desirable to combine administration of the anti-Pro104antibody or antibodies, with administration of an antibody directedagainst another tumor antigen associated with the particular cancer. Assuch, this invention is also directed to an antibody “cocktail”comprising one or more antibodies of this invention and at least oneother antibody which binds another tumor antigen associated with thePro104-expressing tumor cells. The cocktail may also comprise antibodiesthat are directed to other epitopes of Pro104. Preferably the otherantibodies do not interfere with the binding and or internalization ofthe antibodies of this invention.

The antibody therapeutic treatment method of the present invention mayinvolve the combined administration of an anti-Pro104 antibody (orantibodies) and one or more chemotherapeutic agents or growth inhibitoryagents, including co-administration of cocktails of differentchemotherapeutic agents. Chemotherapeutic agents include, e.g.,estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil,melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such aspaclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

The antibody may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (see, EP 616 812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated isandrogen independent cancer, the patient may previously have beensubjected to anti-androgen therapy and, after the cancer becomesandrogen independent, the anti-Pro104 antibody (and optionally otheragents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy, before, simultaneouslywith, or post antibody therapy. Suitable dosages for any of the aboveco-administered agents are those presently used and may be lowered dueto the combined action (synergy) of the agent and anti-Pro104 antibody.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Preferably, the antibody isadministered by intravenous infusion or by subcutaneous injections.Depending on the type and severity of the disease, about 1 pg/kg toabout 50 mg/kg body weight (e.g. about 0.1-15 mg/kg/dose) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A dosing regimen can comprise administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the anti-Pro104 antibody. However, other dosageregimens may be useful. A typical daily dosage might range from about 1pg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of this therapy can be readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of a nucleic acid molecule encoding theantibody is encompassed by the expression “administering atherapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy togenerate intracellular antibodies.

There are two major approaches to introducing the nucleic acid molecule(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid molecule is injecteddirectly into the patient, usually at the site where the antibody isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid molecule is introduced into these isolated cells and themodified cells are administered to the patient either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). Thereare a variety of techniques available for introducing nucleic acidmolecules into viable cells. The techniques vary depending upon whetherthe nucleic acid is transferred into cultured cells in vitro, or in vivoin the cells of the intended host. Techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,the calcium phosphate precipitation method, etc. A commonly used vectorfor ex vivo delivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid molecule transfertechniques include transfection with viral vectors (such as adenovirus,Herpes simplex I virus, or adeno-associated virus) and lipid-basedsystems (useful lipids for lipid-mediated transfer of the gene areDOTMA, DOPE and DC-Chol, for example). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Articles of Manufacture and Kits

The invention also relates to an article of manufacture containingmaterials useful for the detection for Pro104 overexpressing cellsand/or the treatment of Pro104 expressing cancer, in particular breast,ovarian, pancreatic and lung cancer. The article of manufacturecomprises a container and a composition contained therein comprising anantibody of this invention. The composition may further comprise acarrier. The article of manufacture may also comprise a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for detecting Pro104 expressingcells and/or treating a cancer condition and may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Atleast one active agent in the composition is an anti-Pro104 antibody ofthe invention. The label or package insert indicates that thecomposition is used for detecting Pro104 expressing cells and/or fortreating breast, ovarian, pancreatic and lung cancer, or morespecifically ovarian serous adenocarcinoma, breast infiltrating ductalcarcinoma, prostate adenocarcinoma, renal cell carcinomas, colorectaladenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas,and pleural mesothelioma, in a patient in need thereof. The breastcancer may be HER-2 negative or positive breast cancer. The cancersencompass metastatic cancers of any of the preceding, e.g., breast,ovarian, pancreatic and lung cancer metastases. The label or packageinsert may further comprise instructions for administering the antibodycomposition to a cancer patient. Additionally, the article ofmanufacture may further comprise a second container comprising asubstance which detects the antibody of this invention, e.g., a secondantibody which binds to the antibodies of this invention. The substancemay be labeled with a detectable label such as those disclosed herein.The second container may contain e.g., a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forPro104 cell killing assays, for purification or immunoprecipitation ofPro104 from cells or for detecting the presence of Pro104 in a serumsample or detecting the presence of Pro104-expressing cells in a cellsample. For isolation and purification of Pro104, the kit can contain ananti-Pro104 antibody coupled to a solid support, e.g., a tissue cultureplate or beads (e.g., sepharose beads). Kits can be provided whichcontain the antibodies for detection and quantitation of Pro104 invitro, e.g. in an ELISA or a Western blot. As with the article ofmanufacture, the kit comprises a container and a composition containedtherein comprising an antibody of this invention. The kit may furthercomprise a label or package insert on or associated with the container.The kits may comprise additional components, e.g., diluents and buffers,substances which bind to the antibodies of this invention, e.g., asecond antibody which may comprise a label such as those disclosedherein, e.g., a radiolabel, fluorescent label, or enzyme, or the kit mayalso comprise control antibodies. The additional components may bewithin separate containers within the kit. The label or package insertmay provide a description of the composition as well as instructions forthe intended in vitro or diagnostic use.

EXAMPLES Example 1 Production and Isolation of Monoclonal AntibodyProducing Hybridomas

The following MAb/hybridomas of the present invention are describedbelow: Pro104.C1, Pro104.C4, Pro104.C13, Pro104.C17, Pro104.C18,Pro104.C19, Pro104.C24, Pro104.C25, Pro104.C27, Pro104.C34, Pro104.C37,Pro104.C46, Pro104.C48, Pro104.C49, Pro104.C50, Pro104.C53, Pro104.C54,Pro104.C55, Pro104.C57, Pro104.C60, Pro104.C66, Pro104.C75, Pro104.C84,Pro104.D4, Pro104.D6, Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18,Pro104.D19, Pro104.D20, Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31,Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58,Pro104.D62, Pro104.D63, Pro104.D64, Pro104.D68, Pro 104.D69, Pro104.D75,Pro104.D81, Pro104.D85, Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102,Pro104.D106, Pro104.D111, Pro104.D112, Pro104.D113, Pro104.D114,Pro104.D115, Pro104.D116, Pro104.D117, Pro104.D118, Pro104.D119,Pro104.D120, Pro104.D121, Pro104.D122, Pro104.D123, Pro104.D,Pro104.D124, Pro104.D125, Pro104.D126, Pro104.D127, Pro104.D,Pro104.D128, Pro104.D129, Pro104.D130, Pro104.D131, Pro104.D132,Pro104.D133, Pro104.D134, Pro104.D135, Pro104.D136, Pro104.D137,Pro104.D138, Pro104.D139, Pro104.K14, Pro104.K15, Pro104.K16,Pro104.K47, Pro104.K71, Pro104.K72, Pro104.K74, Pro104.K75, Pro104.K76,Pro104.K78, Pro104.K81, Pro104.K87, Pro104.K88, Pro104.K89, Pro104.K155,Pro104.K156, Pro104.K157, Pro104.K158, Pro104.K159, Pro104.K160,Pro104.K163, Pro104.K164, Pro104.K176; Pro104.K217, Pro104.K226,Pro104.K227, Pro104.K240, Pro104.K274, Pro104.K264, Pro104.K281,Pro104.K358 or Pro104.K362.

If the MAb has been cloned, it will get the nomenclature “X.1,” e.g.,the first clone of A7 will be referred to as A7.1, the second clone ofA7 will be referred to as A7.2, etc. For the purposes of this invention,a reference to A7 will include all clones, e.g., A7.1, A7.2, etc.

Immunogens and Antigens Recombinant Proteins, HA & His Tags &Transfected Cells

Pro104 (Testisin) Full Length, Fragment E. coli Expressed Sequence &Protein Production

For immunization of mice and production of the C series of MAbs, aPro104 construct encoding a region of Pro104 from Lys20 to Trp297 wasintroduced into a standard E. coli expression vector via restrictionenzyme sites. The construct was cloned in-frame to the C-terminus of asix-histidine tag so that the Pro104 construct would be expressed as asix-histidine tagged protein of 288 amino acids. The recombinant plasmidwas used to transform competent E. coli cells and Pro104 expression wasperformed in shaker flasks. The bacterial paste, collected afterinduction with IPTG, was used for Pro104 purification via Ni-NTA columnchromatography. Pro104 (Lys20 (underlined)-Trp297) expressed amino acidsequence (the bold type represents the hexa histidine tag) (SEQ IDNO. 1) MAKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWLEHHHHHH

Pro104 (Testisin) Insect Cell Expressed Sequence & Protein Production

For immunization of mice and production of the D series of MAbs, aPro104 construct encoding a honey bee melletin secretion signal, aregion of Pro104 from Ile42 to Trp297 and a six histidine tag was clonedand expressed using standard techniques. Pro104 was purified usingNi-NTA resin. Pro104 (Ile42-Trp297) expressed amino acid sequence(underlined portion represents the honey bee melletin secretion signaland the bold type represents the hexa histidine tag) (SEQ ID NO. 2)MKFLVNVALVFMVVYISYIYADPMAIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWHHHHHH

Pro104 (Testisin) 293T & LMTK Cell Expressed Sequences & ProteinProduction

For screening of both C and D series Pro104 MAbs, full length Pro104protein (Met1-Val314) was expressed both with and without an HA tag(bold) and spacer (underlined) located at the C-terminus, downstreamfrom the recombination site (italics), using standard mammalianexpression techniques. Pro104 transfected human 293T and mouse LMTK cellamino acid sequence (SEQ ID NO. 3)MGARGALLLALLLARAGLRKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFWEWIQKLMAQSGMSQPDPSWPLLFFPLLWALPLLGPVDPAFLYKVVR SRMAS YPYDVPDYASL

Pro104 (Testisin) CHO Cell Expressed Sequences & Protein Production

For immunization of mice and production of the K-series Pro104 MAbs,full length Pro104 protein (Met1-Val314) was expressed without a tagusing standard mammalian expression techniques.

Hamster CHO cells were stably transfected with Tetracycline Receptor(TR) using standard recombinant techniques. Prior to Pro104transfection, CHO-TR cells were cultured in HAM F12 medium with 10%fetal bovine serum (FBS). A vector encoding full length Pro104 protein(Met1-Val314) was transfected into the CHO cells. Stable transfectantswere selected in HAM F12 medium with 10% FBS with Hydromycin B at 300ug/ml, for 15 days. Hydromycin B-resistant cells were checked forexpression of Pro104 by western blot using diadexus Pro104.C25.1monoclonal antibody after 16-20 hour stimulation with 1 ug/mlTetracycline. Cells were expanded, scaled-up and cryopreserved in FBSwith 10% DMSO and stored in liquid nitrogen at −196° C. to assuremaintenance of viable clone cultures. Pro104 transfected hamster CHOcell amino acid sequence (SEQ ID NO. 4)MGARGALLLALLLARAGLRKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSGGPLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWPLL FFPLLWALPLLGPV

Ovr115 Serine Protease Domain Sequence & Protein Production

Ovr115 (TWRSS4) was used to screen out cross reactive hybridoma clones,since this antigen was also upregulated in ovarian and pancreaticcancers and since it also contained a potentially cross-reactive serineprotease domain.

An Ovr115 construct encoding a tobacco etch virus protease (TEV)recognition site and the serine protease domain of Ovr115 from Val203 toLeu435 was cloned in-frame to the C-terminus of glutathioneS-transferase (GST) so that the Ovr115 construct was expressed as aGST-fusion protein of 486 amino acids using standard techniques.Purification of Ovr115 was completed via glutathione sepharose column.Ovr115 serine protease domain construct amino acid sequence (GSTsequence is underlined, TEV sequence is in italics and tag sequence isin bold type) (SEQ ID NO. 5)MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRHNQTSLYKKAGF ENLYFQGVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLTAAHCFRKHTDVFNWKVRAGSDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTFSGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQVIDSTRCNADDAYQGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQWHVVGIVSWGYGCGGPSTPGVYTKVSAYLNWIYNVWKAELSNWSHPQFEK

Ovr115 Extracellular Fragment Sequence & Protein Production

An Ovr115 (TMPRSS4) construct encoding a region of Ovr115 from Lys52 toLeu435, which constituted only the predicted extracellular portion ofthe molecule, was cloned with a six-histidine tag immediately downstreamof codon Leu435. Ovr115 was purified using Ni-NTA resin. Ovr115extracellular construct (Ovr115 Lys52 (underlined)-Leu435) amino acidsequence (6 His tag sequence is in bold type) (SEQ ID NO. 6)MKVILDKYYFLCGQPLHFIPRKQLCDGELDCPLGEDEEHCVKSFPEGPAVAVRLSKDRSTLQVLDSATGNWFSACFDNFTEALAETACRQMGYSSKPTFRAVEIGPDQDLDVVEITENSQELRMRNSSGPCLSGSLVSLHCLACGKSLKTPRVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLTAAHCFRKHTDVFNWKVRAGSDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTFSGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQVIDSTRCNADDAYQGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQWHVVGIVSWGYGCGGPSTPGVYTKVSAYLNWIYNVWKAELHHHHHH

Generation of Stable Ovr115 LMTK Mouse Cell Lines

Full length HA-tagged Ovr115 (Met1-Leu435) (underlined) was transfectedinto mouse LMTK cells after cloning into a mammalian expression vectorwith an HA tag. Individual clones were checked for expression of Ovr115by western blot using anti-HA antibody (Covance, Richmond, Calif.),after 1 week in culture. Ovr115 transfected LMTK amino acid sequence(SEQ ID NO. 7) M DPDSDQPLNSLDVKPLRKPRIPMETFRKVGIPIIIALLSLASIIIVVVLIKVILDKYYFLCGQPLHFIPRKQLCDGELDCPLGEDEEHCKSFPEGPAVAVRLSKDRSTLQVLDSATGNWFSACFDNFTEALAETACRQMGYSSKPTFRAVEIGPDQDLDVVEITENSQELRMRNSSGPCLSGSLVSLHCLACGKSLKTPRVVGGEEASVDSWPWQVSIQYDKQHVCGGSILDPHWVLTAAHCFRKHTDVFNWKVRAGSDKLGSFPSLAVAKIIIIEFNPMYPKDNDIALMKLQFPLTFSGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQVIDSTRCNADDAYQGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQWHVVGIVSWGYGCGGPSTPGVYTKVSAYLNWIYNVWKAEL DPAFLYKVVRSRMASY PYDVPDYASLImmunizations

For generation of the C series MAbs mice were immunized with soluble E.coli expressed Pro104 recombinant protein, encoding a region of Pro104from Lys20 to Trp297 of the full length protein. Groups of 8 BALB/c micewere immunized intradermally in both rear footpads. All injections were25 uL per foot. The first injection (day 1) of 10 ug of antigen permouse was in Dulbecco's phosphate buffered saline (DPBS) mixed in equalvolume to volume ratio with Titermax gold adjuvant (Sigma, Saint Louis,Mo.). Subsequent injections of 10 ug of antigen per mouse occurred ondays 5, 9, 12, 16, 19, 23, 26, 29, 30 and consisted of antigen in 20 uLof DPBS plus 5 uL of Adju-phos adjuvant (Accurate Chemical & ScientificCorp., Westbury, N.Y.) per mouse. The final boost injection on day 33consisted of antigen diluted in DPBS alone. Fusion occurred on Day 37.

For generation of the D series MAbs mice were immunized as above withsoluble insect cell expressed Pro104 recombinant protein, whichcorresponded to a region from Ile42 to Trp297 of the full lengthprotein.

For the K series MAbs mice were immunized with a stably transfected CHOcell line expressing Pro104 on the cell surface. The cell surfaceexpression of Pro104 ranged from 13.3 to 97.0%. In the first twoinjections, the mice were immunized with 1.25×10⁶ cells/mouse, ininjections 3-9, the mice were injected with 3.75×10⁶ cells/mouse. Themice were given a final injection of 2.5×10⁶ cells. Whole cells in HBSS(Hanks Balanced Salt Solution) with no adjuvants were used throughoutthe immunization series. The K series immunization schedule was the sameone used for the C and D series above.

Hybridoma Fusions

Mice were sacrificed at the completion of the immunization protocol anddraining lymph node (popliteal) tissue was collected by steriledissection. Lymph node cells were dispersed using a Tenbroeck tissuegrinder (Wheaton #357426, VWR, Brisbane, Calif.) followed by pressingthrough a sterile 40 uM sieve (VWR) into DMEM and removing T-cells viaanti-CD90 (Thy1.2) coated magnetic beads (Miltenyl Biotech,Baraisch-Gladbach, Germany).

These primary B-cell enriched lymph node cells were then immortalized byelectro-cell fusion (BTX, San Diego, Calif.) with the continuousmyeloma-cell line P3x63Ag8.653 (Kearney, J. F. et al., J. Immunology123: 1548-1550, 1979). Successfully fused cells were selected byculturing in standard Hypoxanthine, Azaserine (HA) (Sigma, St. Louis,Mo.) containing selection medium (DMEM/15% FBS/0.5 ng/mL rIL-6 (Sigma,Saint Louis, Mo.)/10% P388D, (ATCC, Manassas, Va.) conditioned medium).These fusion cultures were immediately distributed, 2 million cells perplate, into wells of 96 well culture plates (Costar Cat. # 3585, VWR).Distributing the culture in 96 well culture plates, immediatelyfollowing fusion, facilitated selection of a larger diversity ofhybridoma clones producing single, specific antibodies. Supernatantsfrom wells were screened by ELISA, for reactivity against Pro104 E. coliexpressed protein, Pro104 insect expressed protein, and for nocross-reactivity with the serine protease Ovr115 extracellular domain(insect expressed).

Monoclonal cultures, consisting of the genetically uniform progeny fromsingle cells, were established after the screening procedure above, bylimiting dilution (Coller, H and Coller, B. Hybridoma 2: 91-6, 1983), orcell sorting of single viable cells into wells of two 96 well plates(VWR), using flow cytometry (Coulter Elite, Beckman Coulter, Miami,Fla.). The resulting murine B-cell hybridoma cultures were expandedusing standard tissue culture techniques. Selected hybridomas werecryopreserved in fetal bovine serum (FBS) with 10% DMSO and stored inLiquid Nitrogen at −196° C. to assure maintenance of viable clonecultures.

Screening & Selection of Antibody Producing Hybridomas

Hybridoma cell lines were selected for production of Pro104 specificantibody by enzyme linked solid phase immunoassay (ELISA). Pro104 orOvr115 proteins were nonspecifically adsorbed to wells of 96 wellpolystyrene EIA plates (VWR). One hundred uL volumes of Pro104 or Ovr115proteins at approximately 1 ug/mL in (DPBS) were incubated overnight at4° C. in wells of 96 well polystyrene EIA plates. Plates were washedtwice with Tris buffered saline with 0.05% Tween 20, pH 7.4 (TBST). Theplate wells were then emptied and nonspecific binding capacity wasblocked by completely filling the assay wells with TBST/0.5% bovineserum albumin (TBST/BSA) and incubating for 30 minutes at roomtemperature (RT). The plate wells were then emptied, 100 uL of hybridomaculture medium samples diluted 1:1 with TBST/BSA was added to the wellsand incubated for 1 hour at RT. The wells were then washed 3 times with(TBS. One hundred uL of alkaline phosphatase conjugated goat anti-mouseIgG (Fc) (Pierce Chemical Co., Rockford, Ill.), diluted 1:5000 inTBST/BSA, was then added to each well and incubated for 1 hour at RT.The wells were then washed 3 times with TBST. One hundred uL of alkalinephosphatase substrate para-nitrophenylphosphate (pNPP) (Sigma, SaintLouis, Mo.) at 1 mg/mL in 1 M Diethanolamine buffer pH 8.9 (Pierce,Rockford, Ill.) was then added to each well and incubated for 20 min. atRT. Color development was stopped by addition of 50 uL of 2N NaOH/well.Bound alkaline phosphatase activity was indicated by the development ofa visible yellow color. The enzymatic reaction was quantified bymeasuring the solution's absorbance at 405 nm wavelength. Culturesproducing the highest absorbance values were chosen for expansion andfurther evaluation. Selected ELISA positive cultures from the original96 well plates were transferred to new 96 well tissue culture plates(VWR).

ELISA Screening of Pro104 MAbs

Hybridomas were retested to confirm continued production of Pro104specific MAbs. Hybridoma cultures with supernatants producing ELISAabsorbance values greater than 1.0 with Pro104 and less than 0.2 withOvr115 were expanded in tissue culture and cryopreserved, as describedabove. Selected Pro104 specific cultures were subcloned by limitingdilution or single cell sorting (Coulter Elite) to ensure geneticallystable and uniform progeny.

Results from ELISA Screening of Cloned Pro104 MAbs

Clones Pro104.C13, Pro104.C18, Pro104.C25 and Pro104.C55 from the firstimmunization with E. coli expressed Pro104 were positive by ELISA withE. coli expressed Pro104, insect expressed Pro104, human 293T cellexpressed Pro104 with and without the HA tag (Table 1 below) and did notreact with the other human serine proteases pancreatic trypsin, lungtryptase and kallikrein (Cal Biochem, San Diego, Calif.) nor plasmin orurokinase (American Diagnostics, Greenwich, Conn.). Pro104.C13,Pro104.C18, Pro104.C19, Pro104.C25 and Pro104.C55 were subcloned andscaled up for further characterization by western blot,immunohistochemistry and immunofluorescence. MAbs Pro104.C37 andPro104.C48 cross-reacted with human urokinase and were not evaluatedfurther by immunohistochemistry or immunofluorescence.

Clones Pro104.D4, Pro104.D6, Pro104.D9, Pro104.D14, Pro104.D18,Pro104.D19, Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51, Pro104.D56,Pro104.D58, Pro104.D64, Pro104.D81, Pro104.D88, Pro104.D91 and Pro04.D94from immunization with the insect expressed Pro104, were positive byELISA with E. coli expressed Pro104, insect expressed Pro104, human 293Tcell expressed Pro104 with and without the HA tag (Table 2) and did notreact with the other human serine proteases pancreatic trypsin, lungtryptase, kallikrein, plasmin nor urokinase, see Table 2 below. ClonesPro104.D12, Pro104.D15, Pro104.D55, Pro104.D62, Pro104.D68 andPro104.D106 were positive by ELISA with E. coli expressed Pro104, insectexpressed Pro104 and were more weakly reactive (ELISA OD 405 nm from0.3-0.8) with the human 293T cell expressed Pro104 (data not shown), butdid not react with pancreatic trypsin, lung tryptase and kallikrein,plasmin or urokinase (Table 2). Pro104.D20 and Pro104.D21 were positiveonly with mammalian (293T) Pro104 protein. Pro104.D4, Pro104.D6,Pro104.D9, Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20,Pro104.D21, Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43, Pro104.D47,Pro104.D51, Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63,Pro104.D64, Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85,Pro104.D88, Pro104.D91, Pro104.D94, Pro104.D102 and Pro104.D106 weresubcloned and scaled up for further characterization by western blot,immunohistochemistry and immunofluorescence. Pro104 MAbs cross-reactingwith other human serine proteases (Pro104.D29 & Pro104.D31) were notevaluated further by immunohistochemistry or immunofluorescence.

FACS Screening for Cell Surface Binding of Pro104 C-Series MAbs

CAOV3 (RT-PCR positive for Pro104) and SKOV3 (RT-PCR negative forPro104) ovarian carcinoma cell lines (ATCC) were grown in DMEM/10% FBS.Prior to staining, the cells were washed once with 10 ml Ca⁺²/Mg⁺² freeDPBS and then 7 ml of warm (37° C.) Cellstripper (Mediatech, Herndon,Va.) was added per 150 cm² flask. The cells were then incubated for 5min. at 37° C. with tapping of the flask to remove tightly attachedcells. The cells were removed and pipetted several times to breakaggregates, then immediately placed in DMEM/10% FBS. The cells were thencentrifuged for 5 minutes at 1300 rpm and resuspended in DMEM/10% FBS.The cells were then incubated at 37° C. for a 30 min. recovery period.Prior to staining, viability of the cells was measured using GuavaViacount (Guava Cytometers, Hayward, Calif.) and if >90% viable theywere distributed into 96-well v-bottom plates (VWR) for staining withMAbs.

Cells were aliquoted at 0.5-1.0×10⁶ cells/well in 96-well v-bottomplates and centrifuged for 2 minutes at 1500 rpm. Supernatants wereaspirated and plates briefly shaken on a vortex mixer to resuspend thecells, then a 200 ul volume of DPBS/3% FBS/0.01% Na Azide (FACS buffer)was added to each well. Centrifugation and aspiration was repeated, then25 uL volumes of hybridoma supernatant or purified MAb were added to thecells. Plates were vortexed to resuspend cells, stored on ice for 15min., then washed in 200 uL of FACS buffer and centrifuged as above.This washing procedure was repeated a twice and then 25 uL volumes ofphycoerythrin (PE) conjugated donkey anti-mouse IgG Fc antibody (JacksonImmunoresearch Laboratories Inc., West Grove, Pa.) were added to cells.After 15 minutes on ice the cells were washed twice, as above and thenresuspended in 250 uL of FACS buffer for analysis on the cell sorter orflow cytometer. In certain cases, for storage overnight at 4° C. priorto analysis, 133 ul volumes of FACS buffer and 67 uL of 1%paraformaldehyde/DPBS were added to wells, for fixation, then thevolumes were increased to 250 uL with DPBS. Stained cells were analyzedon an Eli fluorescent activated cell sorter (FACS) (Beckman-Coulter,Miami, Fla.).

Results demonstrating cell surface binding of several of the C seriesMAbs by immunofluorescent FACS and microscopic analysis, are summarizedin Table 1. Further immunofluorescence microscopy data with human tumorcell lines are presented below. The isotypes of the C series MAbs weredetermined using commercially available mouse monoclonal antibodyisotyping immunoassay test kits (IsoStrip, Roche Diagnostic Corp.,Indianapolis, Ind.). Results of the isotyping are listed in Table 1.TABLE 1 ISOTYPE, ELISA, IMMUNOFLUORESCENCE FACS & MICROSCOPY RESULTS OFPro104 C SERIES MAbs Microscopy Co- Direct ELISA localization Pur. E.coli Pur. Insect FL FACS with HA on Pro104 expressed insect Pro104Pro104 Pro104- CaOv-3 SkOv-3 Pro104-HA MAb Full Lgth expressed Crude293T HA 293T (RT- (RT- Transfected Clone Isotype Pro104 Pro104 LysateLysate Lysate PCR+) PCR−) 293T cells C4 IgG1 k + + + + + − −  4* C13IgG1 k + + − + + + − 3 C18 IgG1 k + + + + + − − 3 C19 IgG1 k + + + − − −− 4 C25 IgG1 k + + + + + + + 3 C34 IgG1 k + + + − − − − 4 C37 IgG1k + + + + + + 3 C46 IgG2b k + + − − − − − 2 C48 IgG1 k + + − + + + − 3C50 IgG2b k + + − − − − − 3 C53 IgG2b k + + − − − − − 3 C54 IgG2bk + + + − − − − 4 C55 IgG1 k + + + + − − − 4 C57 IgG2b k + + − − − + − 2C60 IgG1 k + + + − − + − 4 C66 IgG1 k + + − − − − − 1 C84 IgG1 k +− + + + − − 1 C1 IgG2b k + − − − − + + 2 C17 IgG2b k + − − − − + + 2 C24IgG2a k + − − − − + − 2 C27 IgG3 k + − − − − + − 3 C49 IgG2a k + + − −− + − 2 C75 IgG3 k + + − − − + − 3*4 = Strong co-localization with HA with no background staining ofnon-transfected 293T cells, 3 = Strong co-localization with HA &background staining of non-transfected 293T cells, 2 = Partialco-localization with HA & background staining of non-transfected 293Tcells, 1 = Weak HA staining (possibly blocked by test MAb) & highbackground staining of non-transfected 293T cells.

TABLE 2 RESULTS OF ELISA SCREENING OF THE Pro104 D SERIES MAbs Pro104Pro104 MAb Pro104 Pro104 Ovr115 293T Clone (Insect) (E. coli) LMTKUrokinase Plasmin Tryptase Kallikrein Trypsin lysate D4 + + − − − − −− + D6 + + − − − − − − + D9 + + − − − − − − + D12 + + − − − − − − +D14 + + − − − − − − + D15 + + − − − − − − + D18 + + − − − − − − +D19 + + − − − − − − + D20 − − − − − − − − + D21 − − − − − − − − +D26 + + − + D29 + − − − + − − − + D31 + + − − + − − − + D43 + + − +D47 + + − + D51 + + − + D55 + − − − − − − − + D56 + + − + D58 + + − − −− − − + D62 + + − − − − − − + D63 + + − + D64 + + − + D68 + + − − − − −− + D69 + + − + D75 − − − + D81 + + − + D85 + + − + D88 + + − − − − −− + D91 + + − + D94 + + − + D102 + + − + D106 + + − − − − − − +FACS Screening for Cell Surface Binding of Pro104 D-Series and K-SeriesMAbs

Fifty million 293F cells were transfected by a preparation of lipid-DNAcomplexes by performing a dilution of 50 μg of plasmid DNA in Opti-MEM Ireduced serum medium (GIBCO) to a total volume of 1.5 ml followed bygentle mixing. A dilution of 75 μl of 293Fectin (Invitrogen) in Opti-MEMI to a total volume of 1.5 ml was mixed gently and incubated for 5minutes at room temperature. After the 5 minute incubation, the dilutedDNA was mixed with the diluted 293fectin. This mixture was allowed toincubate for 20-30 minutes at room temperature to allow theDNA-293fectin complexes to form. While the DNA-293fectin complexesincubated, an aliquot of 50 ml of cell suspension (1E6 viable cells/ml)was placed into a sterile, disposable flask. After the DNA-293fectincomplex incubation was completed, they were transferred to each flask ofcells. The flasks were incubated in a 37° C. incubator with shaking at120 rpm. The cells were used for staining experiments at approximately48 hours post-transfection.

The DNA sequence used for transfecting the 293F cells was the fulllength. Pro104 sequence (met¹ to val³¹⁴ with no tags, See SEQ ID NO: 3above.

Prior to staining, the viability of the 293F and control cells wasmeasured using Guava Viacount (Guava Technologies, Hayward, Calif.) andif >90% were viable they were distributed into 96-well v-bottom plates(VWR) for staining with MAbs. Cells were aliquoted at 0.5-1.0×10⁶cells/well in 96-well v-bottom plates and centrifuged for 2 minutes at1500 rpm. Supernatants were aspirated and plates briefly shaken on avortex mixer to resuspend the cells, then 200 ul of DPBS/3% FBS/0.01% NaAzide (FACS buffer) was added to each well. Centrifugation andaspiration was repeated, then 25 uL of hybridoma supernatant or 1ug/million cells of purified MAb was added to the cells. Plates werestored on ice for 15 min., then washed and centrifuged as above, in 200uL of FACS buffer. This washing procedure was repeated twice and then 25uL of goat anti-mouse Ig (H+L) biotin conjugated antibody (CaltagLaboratories, Burlingame, Calif.) was added to the wells for 15 minutesand washed as above. 25 ul of phycoerythrin Streptavidin (PESA) wasadded to cells. After 15 minutes on ice the cells were washed twice, asabove and then resuspended in 250 uL of FACS buffer for analysis on thecell sorter or flow cytometer. Stained cells were analyzed on an Elitefluorescence activated cell sorter (Beckman-Coulter, Miami, Fla.).

Results demonstrating cell surface binding of many of the D-series andK-series MAbs by FACS analysis, are listed in Tables 3A, 3B, and 3Cbelow. Results of representative experiments demonstrating cell surfaceexpression by FACS analysis are depicted in FIG. 1 (A and B) and FIG. 2(A and B).

Specifically, FIG. 1A demonstrates cell surface binding of thePro104.D116.1 antibody to transiently transfected 293F cells compared toa control antibody (Ovr110.A57.1). FIG. 1B indicates the bindingobserved in FIG. 1A is specific to Pro104. In addition, FIG. 2Ademonstrates cell surface binding of the Pro104.D118.1 antibody totransiently transfected 293F cells compared to a control antibody(Ovr110.A57.1). FIG. 2B indicates the binding observed in FIG. 2A isspecific to Pro104.

Binding of the MAb Pro104.D116.1 resulted in 85% of Pro104 transfectedhuman 293F cells being positive, with a MFI (mean fluorescenceintensity) 9-fold higher than cells stained with a control antibody(Ovr110.A57.1) alone. Binding of Pro104.D118.1 resulted in a bimodaldistribution with 70% of the cells being positive for Pro104 and a meanfluorescence intensity 22-fold higher than the control antibody.

The other D-series antibodies that bound significantly to Pro04-293Ftransfected cells and not to untransfected 293F cells were Pro104.D9.1,Pro104.D112.1, Pro104.D113.1, Pro104.D114.1, Pro104.D115.1,Pro104.D119.1, Pro104.D120.1, Pro104.D121.1, Pro104.D122.1,Pro104.D123.1, Pro104.D124.1, Pro104.D125.1, Pro104.D126.1,Pro104.D127.1, Pro104.D129.1, Pro104.D130.1, Pro104.D131.1,Pro104.D132.1, Pro104.D133.1, Pro104.D134.1, Pro104.D135.1,Pro104.D136.1, Pro104.D137.1, Pro104.D138.1, and Pro104.D139.1 (seetable 3A below). TABLE 3A Cell Surface Binding of Pro104 D-Series MAbsto Pro104 Transfected 293F Cells Pro104- Untransfected Transfected 293F293F Cells Cells % Cells % Cells Sample Positive MFI Positive MFI NoStain 1.6 0.429 1.5 0.465 GAMBio SAPE 4.5 0.421 1.7 0.538 SAPE alone 3.80.411 1.4 0.511 Ovr110.A57.1 3.1 0.372 1.6 0.504 (negative control)5E9C11 (positive 80.7 25 99.2 18.1 control) Pro104.D9.1 38 2.63 9.20.802 Pro104.D111.1 5.2 0.593 1.2 0.479 Pro104.D112.1 28.7 2.38 1.40.515 Pro104.D113.1 68.7 6.2 1.8 0.571 Pro104.D114.1 51.5 4.18 1.7 0.509Pro104.D115.1 28.3 2.25 3.9 0.672 Pro104.D116.1 84.8 3.88 1.9 0.594Pro104.D117.1 1.5 2.04 1.2 0.524 Pro104.D118.1 69.9 9.01 1.6 0.524Pro104.D119.1 83.4 3.96 1.2 0.503 Pro104.D120.1 36.4 2.68 1.8 0.531Pro104.D121.1 76.3 7.58 1.9 0.545 Pro104.D122.1 21.3 2.41 1.3 0.513Pro104.D123.1 63 3.12 1.3 0.475 Pro104.D124.1 74.5 3.41 2 0.595Pro104.D125.1 65.1 6.62 3 0.609 Pro104.D126.1 62.5 3.16 1.5 0.483Pro104.D127.1 66.3 3.39 1.4 0.514 Pro104.D128.1 1.9 2.06 1.3 0.504Pro104.D129.1 72.5 3.33 2.9 0.59 Pro104.D130.1 56.7 6.26 3.7 0.65Pro104.D131.1 31.2 2.58 9.7 0.822 Pro104.D132.1 61.2 3.1 2.4 0.614Pro104.D133.1 55 2.99 2.1 0.511 Pro104.D134.1 14.2 2.19 2.3 0.561Pro104.D135.1 53.3 5.87 2.7 0.538 Pro104.D136.1 40.2 2.52 2.3 0.52Pro104.D137.1 56.3 5.86 2.9 0.646 Pro104.D138.1 68.3 6.23 3.1 0.624Pro104.D139.1 59.4 5.98 2.7 0.633

Pro104.K81 (from the K series) antibody also bound to 293F transientlytransfected with Pro104. Approximately 54% of the cells were positivewith a mean fluorescence intensity of 1.69 which was 3-fold over thenegative control antibody.

The other K-series antibodies that bound significantly to Pro104-293Ftransfected cells and not to untransfected 293F cells were Pro104.K72,Pro104.K78, Pro104.K81, Pro104.K88, Pro104.K156, Pro104.K159,Pro104.K164 and Pro104.K176 (see table 3B below). TABLE 3B Cell SurfaceBinding of Pro104 K-Series MAbs to Pro104 Transfected 293F Cells Pro104-Untransfected Transfected 293F 293F Cells Cells % Cells % Cells SamplePositive MFI Positive MFI No Stain 1.1 0.41 0.8 0.456 GAMBio SAPE 1.80.428 1.5 0.482 SAPE 1.4 0.397 0.8 0.459 Pro104.D9.1 70.3 3 1.7 0.482Cln242.B53.1 3.6 0.544 1.4 0.535 (negative control) Pro104.K15 18.10.919 3.2 0.557 Pro104.K47 16.2 0.901 11.7 0.824 Pro104.K71 18.1 0.9631.8 0.479 Pro104.K72 19.7 1.03 1.5 0.475 Pro104.K75 99.2 15.5 13.4 0.816Pro104.K78 99 17.2 2.7 0.495 Pro104.K81 54.2 1.69 3.1 0.56 Pro104.K8855.5 1.95 1.9 0.51 Pro104.K156 98.7 18.7 7.7 0.907 Pro104.K159 39.1 1.341.9 0.488 Pro104.K164 81.8 3.75 4.2 0.581 Pro104.K176 81.1 3.23 2.40.546

Pro104 D-series and K-series antibodies were also tested on cell linesthat were QPCR positive for Pro104 transcript (HeLa) and QPCR negativefor Pro104 transcript (HCT116). Pro104.K81 bound to 97% of HeLa and to9% of HCT116 cells, with a 8-fold higher shift in mean fluorescenceintensity. (See table 3C below). TABLE 3C Cell Surface Binding of Pro104D-Series and K-Series MAbs to Pro104 QPCR Positive Cell Line HeLa HelaCells HCT116 Cells % Cells % Cells Sample Positive MFI Positive MFI NoStain 1.3 0.339 1.6 0.332 GAMBio SAPE 1.4 0.38 1.25 0.48 SAPE 1.3 0.3331.4 0.423 Ovr110.A57.1 1.3 0.375 1.3 0.522 (negative control) anti-CD7199.8 49.6 99.9 38.9 (positive control) Pro104.D4.1 2 0.425 0.7 0.447Pro104.6.1 4.4 0.692 1.5 0.448 Pro104.D9.1 3.3 0.694 0.5 0.408Pro104.D14.1 2.2 0.428 1 0.404 Pro104.D18.1 2.3 0.486 1 0.421Pro104.D19.1 2.1 0.506 1.1 0.421 Pro104.D31.1 6.9 0.634 3.6 0.527Pro104.D58.1 2 0.441 0.5 0.421 Pro104.D116.1 5.5 0.738 0.6 0.428Pro104.D118.1 2 0.478 1.1 0.449 Pro104.D119.1 6.3 0.801 0.8 0.425Pro104.D121.1 4.1 0.656 1 0.432 Pro104.D123.1 3.8 0.696 0.6 0.416Pro104.D124.1 5.3 0.746 0.7 0.418 Pro104.D126.1 4.6 0.711 0.8 0.428Pro104.D132.1 4.6 0.689 1.2 0.431 Pro104.D133.1 5.2 0.706 0.8 0.408Pro104.K14 2.3 0.472 1 0.538 Pro104.K15 92.6 5.84 80.2 3.29 Pro104.K162.8 0.455 1.9 0.505 Pro104.K47 51.8 1.6 69.5 2.09 Pro104.K71 10.9 0.7448.9 0.776 Pro104.K72 10.4 0.769 8.3 0.719 Pro104.K74 1.6 0.442 0.2 0.465Pro104.K75 98.8 14.5 93 7.62 Pro104.K76 2.2 0.42 1.3 0.485 Pro104.K782.7 0.409 1 0.444 Pro104.K81 96.9 6.51 8.9 0.772 Pro104.K87 3.3 0.5313.9 0.641 Pro104.K88 99.3 10.2 96.7 10.1 Pro104.K89 99.6 19.1 43.7 1.34Pro104.K155 1.5 0.413 0.4 0.468 Pro104.K156 2.4 0.491 1.1 0.505Pro104.K157 97.4 10.3 92.9 6.47 Pro104.K158 2.3 0.454 2.4 0.519Pro104.K159 15.8 0.87 22.3 0.908 Pro104.K160 36.7 1.3 29 1.08Pro104.K163 4.1 0.49 13.9 0.751 Pro104.K176 31.7 1.18 47 1.29Pro104.K217 11.9 0.767 25.4 0.949 Pro104.K226 9.6 0.736 11.4 0.75Pro104.K227 42.4 1.42 65.5 2.19 Pro104.K240 97.5 8 95.8 15.2 Pro104.K27423 0.923 15.1 0.622 Pro104.K264 3.6 0.497 1.5 0.53 Pro104.K281 55.2 2.0870.7 3 Pro104.K358 3.8 0.539 4.1 0.625 Pro104.K362 3.9 0.534 3.2 0.61

These results indicate that the antibodies in Tables 3A, 3B and 3C andin particular, Pro104.K81 Mab are suitable for immunotherapy of tumorswith or without conjugated drugs, toxins, enzymes, prodrug activatingmolecules or isotopes.

Pro104 MAb Affinity Analyses

Binding kinetics and affinity constants were calculated from surfaceplasmon resonance measurements using a BIACORE 3000 instrument (Diacore,Piscataway, N.J.). Experiments were designed to simultaneously generateon rate, off rate, and affinity values for the Pro104 MAbs.

Pro104 protein lot# 060402 (diaDexus) was immobilized on flow cell 2 ofa CMS sensor chip (Biacore) by standard amine coupling (Biacore). Flowcell 1 was used as a blank surface for reference subtractions, and wasactivated and then inactivated with ethanolamine. Pro104 MAbs werediluted in HBS EP buffer (Biacore) and passed over flow cells 1 and 2 inseries. MAbs were injected in duplicate, sequentially, for each of fiveconcentrations: 200, 100, 50, 25, 12.5 ug/mL. Assuming a molecularweight for the MAbs of 158,000 kDa, the respective concentrations in nMwere calculated to be: 1266, 633, 317, 158 and 79. MAbs were injectedfor 3 minutes at 30 uL/min followed by a dissociation time of 12minutes. The regeneration of the chip surface, or removal of MAb betweencycles, was performed by passing two injections of 100 mM Glycine pH 1.5through the flow cells firstly for 30 seconds and then for 12 seconds,both at 100 uL/minute.

The above procedure was performed by using the Biacore's kineticanalysis wizard included in the Biacore control software. The resultingsensograms were fitted automatically, assuming a 1:1 Langmuir bindingmodel. The results presented in Table 4 below were calculated using thewizard data processing function. The calculated affinities presented inTable 3 were all in the 10⁻⁹ M range except for Pro104.C34 and hencewere sufficiently high to achieve a therapeutic dose in-vivo at lessthan or equal to 10 mg/kg, excepting for Pro104.C34. TABLE 4 Pro104 CSeries MAb Affinities Pro104 Biacore Affinity CharacterizationAnti-Pro104 Antibody Clone KA KD ka Kd C4 3.5E+08 2.9E−09 2.6E+047.4E−05 C18 3.4E+08 2.9E−09 2.6E+04 7.5E−05 C25 3.5E+08 2.8E−09 3.0E+048.5E−05 C37 5.5E+08 1.8E−09 5.4E+45 9.7E−05 C48 3.6E+08 2.8E−09 4.0E+041.1E−04 C60 1.6E+08 6.4E−09 3.0E+04 1.9E−04 C19 1.9E+08 5.2E−09 3.8E+042.0E−04 C34 3.2E+08 3.0E−05 2.0E+01 6.2E−04 C54 5.5E+08 1.8E−09 3.7E+046.7E−05 C55 1.0E+08 9.8E−09 1.6E+04 1.5E−04Western Blots

Protein extracts for western blot analysis were prepared in cell lysisbuffer (1% NP40/10 mM Sodium Phosphate pH 7.2/150 mM sodium chloride)from Pro104-293T transient transfectants and mammalian adenocarcinomacell lines. Proteins were separated by electrophoresis on NuPAGE 4-12%Bis-Tris gels (Invitrogen Life Technologies, Carlsbad, Calif.) underdenaturing conditions in a Novex-XCell II Minicell gel apparatus(Invitrogen Life Technologies, Carlsbad, Calif.) and subsequentlytransferred to PVDF membranes using an XCell II Blot Module (InvitrogenLife Technologies, Carlsbad, Calif.). Following the transfer ofproteins, the membranes were blocked in 1% blocking reagent (Cat. #1 096176, Roche Diagnostic Corp., Indianapolis, Ind.) and incubated overnightat 4° C. with purified primary antibodies Pro104.C4, Pro104.C13,Pro104.C18, Pro104.C19, Pro104.C25, Pro104.C34, Pro104.C37, Pro104.C48,Pro104.C55, Pro104.C60 or Pro104.C66, and then withhorseradish-peroxidase conjugated goat anti-mouse IgG (qat.#115-036-062, Jackson Immunoresearch Laboratories, Inc.). Bands werevisualized by chemiluminescence using an ECL advance western blottingdetection kit (Cat #RPN2135, Amersham Biosciences, Piscataway, N.J.).

Deglycosylation experiments were performed on protein extracts fromPro104-293T transfectants, mammalian adenocarcinoma cell lines andnormal human testis by treating with peptide N-glycosidase F (PNGaseF,Cat #P0704S, New England Biolabs, Inc, Beverly, Mass.) as directed bythe manufacturer. The deglycosylated samples were then analyzed bywestern blotting as described above. Briefly, 100 ug volumes of proteinextracts were denatured in glycoprotein denaturing buffer (0.5% SDS/1%beta-mercaptoethanol), at 100° C. for 10 minutes. This was followed bythe addition of kit reaction buffers (New England Biolabs) to a finalconcentration of 1% NP-40 and 50 mM sodium phosphate, addition of 100units of PNGase F and incubation at 37° C. for 4 hours. TABLE 5 RESULTSFROM WESTERN BLOTS USING PRO104 MABS WITH EXTRACTS FROM TRANSFECTED 293TCELLS & HUMAN ADENOCARCINOMA CELL LINES HeLa & Pro104 DeglycosylatedCaOv3 Deglycosylated SkOv3 MAb Pro104-293T Pro104-293T (RT-PCR+) HeLa &CaOv3 (RT-PCR−) C4 +Multiple bands 35-40 kDa C13 +Multiple bands 35-40kDa C18 +Multiple bands 35-40 kDa C19 +Multiple bands 35-40 kDa C25+Multiple approx. 30 kDa approx. approx. 30 kDa — bands 35-40 kDa 38 kDaC34 +Multiple bands 35-40 kDa C37 +Multiple approx. — bands 35-40 kDa 38kDa C48 +Multiple bands 35-40 kDa C55 +Multiple approx. — bands 35-40kDa 38 kDa C60 +Multiple bands 35-40 kDa C66 —

Results of the western blot experiments are summarized in Table 5 above.In whole cell lysates from Pro104-293T transfectants, the MAbsPro104.C4, Pro104.C13, Pro104.C18, Pro104.C19, Pro104.C25, Pro104.C34,Pro104.C37, Pro104.C48, Pro104.C55 and Pro104.C60 reacted specificallywith several protein bands from approximately 35 kDa to 40 kDa, whichwere consistent with glycosylated forms of Pro104 formed afterprocessing of full length Pro104/testisin. These bands were absent inthe non-transfected 293T cell line sample. Pro104 MAb-C66 was notreactive by western blot analysis and was therefore eliminated fromfurther studies. A protein eband at approximately 38 kDa was detected byMAbs Pro104.C25, Pro104.C55 and Pro104.C37 in lysates from Pro104 mRNApositive (RT-PCR+) cancer cell lines HeLa and CaOv3 (ATCC), but wasabsent as expected, in lysates from the RT-PCR negative ovarian cancercell line SkOv3 (ATCC). Similarly, Nbs Pro104.C25 and Pro104.C55detected a band of the predicted molecular weight (38 kDa), in lysatefrom normal human testis (data not shown). MAbs Pro104.C25 andPro104.C55 also reacted with recombinant and native mouse testisin (datanot shown), in western blots.

In western blots on deglycosylated lysates from Pro104 transfectants,RT-PCR positive cell lines and normal testis, the migration of thePro104 protein as detected by Pro104.C25, shifted from approximately38-40 kDa (glycosylated) to approximately 30 kDa (non-glycosylated).This reduction in molecular weight of Pro104 is consistent with theprediction of three N-glycosylation sites on the catalytic subunit ofPro104 protein.

Example 2 Cell surface binding of Pro104 MAbs in Live Cancer CellsDemonstrated by Immunofluorescence

The following cancer cell lines were used in this study and wereobtained from the ATCC: Cervical (HeLa), Ovarian (Tov-112D, Tov-21G,CaOV-3 and SKOV-3), colon (HCT116) as well as the pancreatic (MIA Paca-2and AsPC). HeLa, CaOV-3 cell lines express Pro104 RNA as determined byQPCR. Control HCT116 and SKOV-3 cells do not express Pro104 RNA.

The above cell lines were seeded onto sterile 18 mm glass coverslips andcultured at 37° C. in DMEM/10% FBS with penicillin and streptomycin for48 hr prior to treatment with the primary antibodies (Pro104 MAbs). MAbsPro104.C19.1, Pro104.C25.1, Pro104.C55.1 and Pro104.D9 were tested byimmunofluorescence microscopy to determine which of these antibodiesbound specifically to the cell surface of Pro104 expressing cancercells. Primary MAbs were added to the medium at a final concentration of10 ug/ml and incubated for one hour at 37° C. Following fixation with 3%formaldehyde in Phosphate Buffered Saline (PBS), the cells wereincubated with a secondary Cy3-labeled donkey anti-mouse (JacksonImmunoresearch Laboratories, West Grove, Pa.) at a concentration of 5ug/ml for 30 min. Following washing, the cells were mounted inVectastain (Vector, Burlingame, Calif.), a medium containing DAPI tovisualize the cell nuclei and observed in a Zeiss Axiophot fluorescencemicroscope (Carl Zeiss, Thornwood, N.Y.) equipped with the appropriatefluorescent filters. Micrographs were recorded using a CCD camera.

Pro104.C19.1, Pro104.C25.1, Pro104.C55.1 and Pro104.D9 all bound toPro104 expressing cells. FIGS. 3A and 3B demonstrate the binding ofPro104.C19.1 to HeLa cells (FIG. 3A). Most of the cells in the fieldshowed labeling for Pro104. Pro104.C19.1 could clearly be seendecorating the cell membrane of the cells (arrows). However,Pro104.C19.1 did not bind to the control negative (QPCR) SKOV-3 cancercells (FIG. 3B, N indicates the position of the cell nuclei).

Binding and Internalization in Live Cancer Cells by Cy3 ConjugatedAntibodies

This study was performed using directly conjugated fluorescentantibodies (MAbs). Using antibodies directly conjugated with thefluorescent dye Cy3, antibody binding and internalization can bevisualized by fluorescence microscopy. This technology is well known inthe art. SKOV-3 cells that do not express Pro104 (QPCR negative) wereused as negative controls.

Cy3 Conjugation

Pro104.C19.1, Pro104.C25.1 and Pro104.A55.1 MAbs were each conjugated toCy3. Cy3 conjugation was carried out according to standard procedures inthe manufacturer's guidelines Pierce). Briefly, 1 mg of antibody wasdialyzed against 0.1M bicarbonate buffer (pH 9.3) for 60 min, mixed withCy3 dye and incubated at RT for 2 hr, then transferred to a Slide-ALyzer Dialysis cassette (Pierce) and dialyzed in 2 liters of PBS for 6hr at 4° C. The dialysis buffer was replaced and dialysis was repeated 6times. The Cy3 conjugated antibodies were recovered and concentrationwas measured in a spectrometer at 280 nm.

Cell Labeling

Cy3-Pro104.C19.1, Cy3-Pro104.C25.1 and Cy3-Pro104.A55.1 MAbs wereincubated with the cells at a concentration of 10 ug/ml at 37° C. inwater chambers for 60 min and then the coverslips with cells were washedin PBS and the cells were fixed with 3% formaldehyde in PBS for 10 min.Following fixation, the coverslips with the cells were mounted in amedium containing DAPI (Vectastain) to visualize cell nuclei and thecells observed using a Zeiss fluorescence Microscope Axiophot equippedwith the appropriate fluorescent filters. Micrographs were obtained witha CCD camera.

Results

Immunofluorescence microscopy of cancer cells treated withCy3-Pro104.C19.1, Cy3-Pro104.C25.1 and Cy3-Pro104.A55.1 indicated thatovarian and pancreatic cancer cells expressing Pro104 were able to bindand internalize the fluorescent antibodies. FIG. 4A shows the binding ofCy3-Pro104.C25.1 to the cell surface of HeLa cells (arrows), a cell linethat expresses Pro104. The Cy3-Pro104.C25.1 antibody did not bind to thecontrol cells SKOV-3 which do not express Pro104. See FIG. 4B, Nindicates the nuclei of several unlabeled cells. FIG. 5 demonstratesthat, following the binding to the cell membrane, Cy3-Pro104.C25.1 wasinternalized in live HeLa cells and that internalization vesicles couldbe observed in the cytoplasm of these cells. In particular, vesiclescould be often visualized in close proximity to the cell nuclei (N)(arrow). FIG. 6A and FIG. 7A show the binding and internalization ofCy3-Pro104.19.1 and Cy3-Pro104.C55.1 in MIA-PaCa-2 cells, respectively.MIA-PaCa-2 cells are a pancreatic cell line that expresses Pro104. Theinternalization pattern was characterized by the presence of perinuclearvesicles likely to correspond to endosomes located in the proximity ofthe Golgi apparatus (arrows). Cy3-Pro104.C19.1 and Cy3-Pro104.C55.1 didnot bind to the cells of the control cell line HCT-116 which does notexpress Pro104 (FIGS. 6B and 7B).

Cy3 conjugated MAbs Pro104.C19.1, Pro104.C25.1 and Pro104.A55.1 were allinternalized upon binding to the cell surface of Pro104 expressingcancer cells, in-vitro. These results indicate anti-Pro104 antibodies,and in particular, Pro104.C19.1, Pro104.C25.1 and Pro104.A55.1 MAbs aresuitable for immunotherapy of tumors with or without conjugated drugs,toxins, enzymes, prodrug activating molecules or isotopes.

Distribution of Pro104 in Tumors and Normal Tissues byImmunohistochemistry (IHC)

Formalin fixed paraffin embedded blocks of ovarian and pancreatic cancerand normal adjacent tissues were obtained from the National DiseaseResearch Interchange (Philadelphia, Pa.). OCT embedded blocks of normalorgans were obtained from Zoion (Hawthorne, N.Y.).

Immunohistochemical Staining for Formalin Fixed Paraffin EmbeddedSections

Six μm thick sections cut from formalin fixed paraffin embedded blockswere heated at 45° C. for 15 min, deparaffinized in Histoclear (NationalDiagnostics, Atlanta, Ga.), deparaffinized in Histoclear and rehydratedthrough a series of reducing ethanol concentrations to PBS. Antigenretrieval was performed by boiling the section slides in 10 mM sodiumcitrate buffer (pH 6.0), at 120° C., 15-17 PSI in a decloaking chamber(Biocare, Walnut Creek, Calif.) for 10 min. Endogenous peroxidaseactivity was quenched by treating the sections with 3% hydrogen peroxidesolution for 15 min. Slides were incubated with 1% BSA to blocknonspecific antibody binding and then reacted with the primary Pro104MAbs used at a concentration of 10 ug/ml for 1 hour at room temperaturein a DAKO autostainer (Dako Co., Carpinteria, Calif.). After washing inTris-Buffered Saline (TBS) with 0.5% Tween-20, slides were thenincubated with anti-mouse IgG conjugated to horse radish peroxidase(HRP) (Immunovision technologies, Daly City, Calif.). After washing inTBS with 0.5% Tween-20, sections were treated by 3,3′-diaminobenzidinechromagen for 2-5 minutes (Immunovision Technologies) and counterstainedwith hematoxylin before mounting in Permount medium (American MasterTech Scientific, Inc, Lodi, Calif.) after dehydration. Normal mouse IgGat the same concentration as the primary antibody, served as negative acontrol for immunolabeling specificity. In additional controlexperiments, the Pro104 MAbs were incubated with the antigen Pro104before being applied to the histological sections.

Immunohistochemical Staining for OCT Embedded Frozen Unfixed Sections

Slides were cut in the cryochamber at 5-8 um at an appropriatetemperature, air dried for a minimum of thirty minutes at roomtemperature. Briefly, slides were rinsed in TBS to remove off OCT andincubated at room temperature. IHC was performed using the ImmunovisionPowervision Kit (Immunovision Technologies, Co. Daly City, Calif.).Briefly, slides were rinsed in TBS-T to remove off OCT and incubatedwith Pro104 primary antibodies for 1 hour at room temperature. They werethen post-fixed in 4% paraformaldehyde fixative for 10 min at roomtemperature and treated as described above.

Results

Pro104.C25.1, Pro104.A55.1, Pro104.D9 and Pro104.D133 were used toimmunolabel sections of ovarian and pancreatic cancer. Epithelial cellsin the ovarian and pancreatic tumors but not in normal ovary andpancreas were labeled. Pro104.C25.1 labeled the cell surface of 10 outof 17 (58%) serous ovarian cancer and 11/11 (100%) pancreatic cancerclinical samples. Pro104.C55.1 labeled the cell surface of 6 out of 8(75%) ovarian cancer and 3/3 (100%) pancreatic cancer clinical samples.Pro104.D9 labeled the cell surface in ¼ ovarian cancer (25%). FIGS. 8A,8B, 8C and 8D illustrate the IHC results obtained with Pro104.C25.1 intwo ovarian cancer clinical samples (FIGS. 8A and 8C). Control normalovaries were not labeled by the Pro104.C25.1 MAb (FIGS. 8B and 8D). FIG.9 shows a higher magnification of an ovarian cancer histological sectionlabeled with Pro104.C25.1. The labeling clearly localized to the cellmembrane of the tumor epithelial cells (arrows).

FIG. 10 demonstrates that Pro104.D9 labeled the cell surface of ovariancancer cells (arrow). Additionally, FIG. 11 shows that Pro104.D133labeled the cell surface of serous ovarian cancer cells.

FIGS. 12A and 12C illustrate the immunolabeling pattern obtained withPro104.C25.1 in clinical samples of pancreatic adenocarcinoma. Pro104labeling was mostly restricted to the cell surface of epithelial cells(arrows) with occasional cytoplasmic labeling (FIG. 12C). Normalpancreatic cells were mostly devoid of specific labeling (FIGS. 12B and12D). Additionally, FIG. 13 shows that no specific labeling was observedwhen normal mouse IgG was used instead of Pro104.C25 (FIG. 13A) or whenPro104.C25.1 was adsorbed with Pro104 antigen prior to processing forIHC (FIG. 13B).

Pro104 expression was also analyzed in normal organs. IHC on OCT frozensections showed no detectable labeling on the cell surface in the cellsof normal heart, liver, kidney, brain, colon, stomach, lung, prostate,ovary, pancreas and breast. However, the membrane of the germ cells inthe testis showed strong Pro104 immunolabeling. This result was expectedfrom data published in the scientific literature (J. D. Hooper et al.Testisin, a new human serine protease expressed by premeiotic testiculargerm cells and lost in testicular germ cell tumors. Cancer Research59:3199-3205 (1999)). See table 6 below for summary. TABLE 6 Summary ofIHC results for Pro104 D-Series MAbs Pro104 D MAb ImmunohistochemistryResults Unfixed OCT Formalin Ovarian Fixed (FFPE) Testis Normal Cancers(no. Ovarian mAb Dilution Germ Smooth vital positive/no. cancers (RatioClone (ug/ml) cell Stroma muscle Other organs* tested) Testispositive/tested) IgG1 10 ug/ml — — — — Anti- 3+ (5/5) 3+ (2/2) KeratinD9  5 ug/ml 3+ — — — — 3+ (2/3) 3+ D116 10 ug/ml 3+ — — — — 1+ (3/5)D119 10 ug/ml 3+ — — — — 1+ (3/5) D121 10 ug/ml 3+ — — — 3+ (all 1+(4/5) 2+ (3/3) 3) D124 10 ug/ml 3+ — — — — 1+ (3/5) D123 10 ug/ml 3+ — —— — 2+ (2/5) 3+ 2+ (2/2) D126 10 ug/ml 3+ — — — +/− 2+ (1/5) 3+ 1+ (2/2)D132 10 ug/ml 3+ — — — — 2+ (1/5) 3+ 2+ (5/5) D133 10 ug/ml 3+ — — — —2+ (1/5) 3+ 2+ (2/2)*Normal vital organs include heart, liver and kidney.

The immunohistochemistry results above demonstrate Pro104 is expressedin a high percentage of ovarian and pancreatic cancer cases. The factthat Pro104 is expressed on the cell surface of cancer cells makes it anideal target for antibody based therapy. Additionally, binding ofanti-Pro104 antibodies to ovarian and pancreatic cancer cellsdemonstrated by IHC indicates anti-Pro104 antibodies, and in particular,Pro104.C25.1, Pro104.D9 and Pro104.D133 MAbs are suitable forimmunotherapy of tumors with or without conjugated drugs, toxins,enzymes, prodrug activating molecules or isotopes.

Example 3 Mouse Monoclonal Sandwich ELISA Detection of Pro104

Pro104 Competitive Checkerboard ELISA

High binding polystyrene plates (Corning Life Sciences (MA)) were coatedovernight at 4° C. with 1 μg/well of anti-Pro104 MAb. The coatingsolution was aspirated off and free binding sites were blocked by adding300 μl/well of Superblock-TBS (Pierce Biotechnology, Illinois) for 1hour at RT. After washing twice with Wash Buffer (1×TBS/0.05% Tween20),100 A1 volumes of Pro104 antigen were added to each well. Each pair wastested with 100 ng/ml and 0 ng/ml of recombinant Pro104 E. coliexpressed protein diluted in Assay Buffer (1×TBS, 1% BSA/1% MouseSerum/1% Calf Serum/0.1% Tween20). After addition, plates were incubatedfor 1 hour at RT with shaking, and washed 4× with 360 μl of Wash Buffer.Then 50 μl volumes of unlabeled coating MAb, at 20 μg/ml in AssayBuffer, were added and incubated with shaking at RT for 10 min.Afterwards, 50 μl volumes of biotinylated detecting MAb (2 μg/1 ml) wereadded to each well and plates were incubated for 1 hour at RT, withshaking. After washing, 100 μl volumes of alkaline phosphataseconjugated streptavidin (Jackson ImmunoResearch Laboratories, PA, 1:2000dilution) were added to wells and plates were incubated for 30 min. atRT, with shaking. After washing, the plate was then developed using pNPPsubstrate in 1×DEA buffer (Pierce Biotechnology, Illinois) for 30 min.at RT. The reaction was stopped by adding 100 μl/well 1N NaOH, andplates were read at 405 nm using a Spectramax 190 plate reader(Molecular Devices, CA). OD readings at 405 nm were used to calculatesignal to noise ratio (OD at 100 ng/mL divided by OD at 0 ng/mL) of eachAb pairs.

The results of the checkerboard ELISA testing 13 MAb are shown in Table7 below. Each antibody was used as a coating as well as a detectingantibody in all possible combinations. All pairs were tested induplicate on 100 and 0 ng of Pro104 E. coli protein in assay buffer(containing mouse serum, calf serum and BSA to be used as blank). Theresults are shown as specific signal/noise (assay buffer alone) ratio.During the incubation with detecting antibody, a 10-fold higherconcentration of coating antibody was added to the wells to preventself-pairing. Self-pairing may be observed when antigens are partlymultimerized and may confound MAb pairing results. Performing the ELISAassay under competitive conditions ensures that antibodies cannot bindto the same or proximal epitopes when the antigen is aggregated.

The data suggest a minimum of five epitopes have been identified sincesteric hindrance may also be a contributing factor to the non-pairing ofMAbs. The epitope map of the Pro104 MAbs derived from the results inTable 7 is shown in FIG. 14. More than 50% of the monoclonal antibodies(Pro104.C4, Pro104.C18, Pro104.C25, Pro104.C37, Pro104.C48 andPro104.C60) reacted with one epitope or epitopes proximal enough tocause steric hindrance and so block MAb pairing in the assay. The MAbsPro104.C34 and Pro104.C13 both reacted with epitopes that were distinctfrom one another and distinct from the other epitopes or MAb groups. TheMAbs Pro104.C55 and Pro104.C19 reacted with an epitope or two proximalepitopes which were sufficiently close to the epitope identified byPro104.C66 to cause partial blocking. The MAbs Pro104.C55 and Pro104.C19also reacted with an epitope or epitopes which were sufficiently closeto the epitope or epitopes identified by the MAb group Pro104.C4,Pro104.C18, Pro104.C25, Pro104.C37, Pro104.C48 and Pro104.C60 to causepartial blocking. However, MAb Pro104.C66 reacted with an epitope whichwas sufficiently distant from the epitope or epitopes identified byPro104.C4, Pro104.C18, Pro104.C25, Pro104.C37, Pro104.C48 and Pro104.C60to allow pairing with MAbs of this group.

Several different MAb combinations were tested to establish a sandwichELISA assay for the detection of native Pro104 from medium or lysates ofcancer cell lines, transfected cell lines and cancer tissues. The pairsPro104.C19/C48 and Pro104.C55/C34 performed best in the Sandwich ELISAwith a sensitivity for recombinant Pro104 at approximately 1 ng/ml.Pro104 was detected by sandwich ELISA in CHAPS (Pierce) detergentlysates from RT-PCR positive CaOV3 ovarian cancer cells, RT-PCR positiveHeLa cervical cancer cells and in lysates from Pro104 transfected 293Tand LMTK cells, 48 hours post transfection. Pro104 was not detected inlysates from the prostate cancer cell line PC3 (ATCC) nor the coloncancer line HT29 (ATCC), which are Pro104 negative by RT-PCR. Theseresults were also in agreement with immunofluorescence data. However,Pro104 protein could not be detected in the tissue culture medium fromany of these cancer cell lines, or in the medium from Pro104 transfectedcells, at 48 hours post transfection. TABLE 7 Identification of Pro104ELISA MAb Pairs by Competition ELISA Detecting Antibody C4 C13 C18 C25C37 C48 C60 C66 C19 C34 C55 Coating Antibody C4 4.3* 50 4 5.3 4.7 5.12.1 12 C13 33 5.3 40 32 26.7 32 28 8.3 17 9 16 C18 3.2 43 2.7 3.1 3.63.9 1.54 10 C25 4 47 4.3 5 5.6 5.1 1.67 1.3 C37 4.2 46 4 4.3 4.4 4.9 2.313 C48 3 50 3.7 3.9 3.7 3.8 1.9 14.7 1.3 19.5 1 C60 22 46 21 19 18 254.38 7.8 C66 31 30 34 23 19 22 13 2 12 11 10 B8 22.8 22.4 21/40 16 13 177.8 2.6 26 15.6 27 B11 17 14 9.7 7.9 11 5.3 1.4 C19 34 33 34 14 1.6 3111 C34 8.7 7.7 5.6 1.9 4.3 1.4 4.3 C55 38 7.8 13.4 13.6 3 27 2.3*Signal to noise (OD at 100 ng/mL divided by OD at 0 ng/mL (assay bufferalone)) ratio.

Example 4 Detection of Pro104 Protein and Phosphorylation of EGFReceptor

Detection of Pro104 Protein in Cell Lines and Ovarian Tumors by WesternBlot

Rk3E, HeLa, ASPC1 and HT29 cells lines were evaluated for expression ofPro104. The RK3E and HT29 cell lines are negative for Pro104 mRNA. As acontrol RK3E was transfected with Pro104 (RK3E-104) using methods knownin the art. As an additional control RK3E cells were also transfectedwith Alkaline Phosphatase (RK3E-AP). HeLa and AsPC1 are positive forPro104 mRNA. In addition to the cell lines, ovarian tumor and normaladjacent tissue to the tumor was evaluated for the presence of Pro104.

Cell extracts were prepared on ice using modified RIPA buffer (1% NP40,10 mM Na₂PO₄, 0.15M NaCl) plus a protease inhibitor cocktail (RocheInc.). Between 20 and 50 ug of protein extract were used for each gellane; protein equivalent concentrations were evaluated for protein levelcomparisons on the same gel. Clarified extracts were mixed with an equalvolume of 2× concentrated Laemmli sample buffer (Invitrogen LifeTechnologies, Carlsbad, Calif.), heated to 70° C. for 10 minutes andthen analyzed using pre-cast 4-12% SDS-polyacrylamide minigels (Nupage;Invitrogen Life Technologies Carlsbad, Calif.) with MES running buffer(Nupage; Invitrogen Life Technologies, Carlsbad, Calif.). Gels weretransferred to Immobilon-P PVDF membranes with a 0.45 μm pore size(Invitrogen Life Technologies, Carlsbad, Calif.) using 1× Nupagetransfer buffer plus 10% Methanol. The membranes were rinsed and blockedfor 1 hour at room temperature using 5% nonfat dry milk in PBS with0.05% Tween-20. Membranes were incubated with primary antibody overnightin 5% nonfat dry milk in PBS with 0.05% Tween-20. A mouse monoclonalantibody directed against Pro104 was produced in house using recombinantbacterial Pro104 protein. The Pro104 monoclonal antibody was diluted1:1000 for a final concentration of 1 ug/ml and a mouse monoclonalantibody against GAPDH (Chemicon Inc., Temecula, Calif.) was diluted1:5000 (for a final concentration of 0.2 ug/ml). Following primaryantibody incubation, membranes were washed four times at roomtemperature for 10 min. each in 1×PBS with 0.05% Tween-20. Horseradishperoxidase linked goat anti-mouse immunoglobulin (Jackson Lab Inc., BarHarbor, Me.) was used (1:10,000 dilution) in 5% nonfat dry milk in PBSplus 0.05% Tween-20 for 1 hour at room temperature to detect the primarymonoclonal antibody. Membranes were finally washed four times for 10min. in 1×PBS plus 0.05% Tween-20 followed by detection using enhancedchemiluminescence (ECL) reagent per manufacturer's directions (Amersham,Piscataway, N.J.) and exposure to X-ray film (Kodak, Rochester, N.Y.).For the Western immunoblot experiment comparing RK3E cells infected withan AP(alkaline phosphatase)-expressing retrovirus with the same cellsinfected with a Pro104-expressing retrovirus, cells were plated ingrowth medium containing either 1% or 10% FBS for 48 hours. Cellextracts were prepared using modified RIPA buffer including aphosphatase inhibitor cocktail (Calbiochem) and 25 ug of clarifiedextract were evaluated by SDS-PAGE and Western immunoblot with apolyclonal antibody specific for the phosphorylated EGF receptor(BioSource International, Camarillo, Calif.).

FIG. 15A demonstrates by western blot Pro104 protein was detected inPro104 transfected cells lines (RK3E-104) and cell lines nativelyexpressing Pro104 (HeLa and AsPC1). Pro104 protein was not detected inAP transfected cell lines (Rk3E-AP) and mRNA negative cell lines (HT29).Additionally, FIG. 15B illustrated detection of Pro104 protein bywestern blot in ovarian tumor tissues but not in normal adjacenttissues.

The fact that Pro104 is detectable in cancer cell lines and ovariantumor tissue makes it an ideal target for antibody-based therapy.Anti-Pro104 antibodies are suitable for immunotherapy of tumors with orwithout conjugated drugs, toxins, enzymes, prodrug activating moleculesor isotopes.

Phosphorylation of EGF Receptor

RK3E transfected cell lines overexpressing Pro104 (RK3E-Pro104) wereevaluated for phosphorylation of the Epidermal Growth Factor (EGF)Receptor. As a control RK3E cells were also transfected with AlkalinePhosphatase (RK3E-AP). Using methods known in the art, phosphorylationof the EGF receptor was evaluated with 10% and 1% serum from RK3E-Pro104and RK3E-AP cells.

Over expression of Pro104 was found lead to phosphorylation of EGFreceptor. FIG. 16 is a western immunoblot against phosphorylated EGFReceptor which demonstrates EGF receptor is phosphorylated fromoverexpression of Pro104 compared to AP controls.

Example 5 Glycosylation, GPI-Linkage and Biotinylation of Pro104 Protein

Pro104 Glycosylation

Deglycosylation experiments were performed on protein extracts from HeLacell lines (Pro104 mRNA positive) and ovarian cancer tumor samples usingPeptide N-Glycosidase F (PNGaseF, Cat#P0704S, New England Biolabs, Inc,Beverly, Mass.) as per the directions provided by the manufacturer. Thedeglycosylated samples were then analyzed by western blotting asdescribed above. Briefly, 10 ug of protein extract was denatured inglycoprotein denaturing buffer/0.5% SDS/1% beta-mercaptoethanol, at 100°C. for 10 minutes. This was followed by the addition of kit reactionbuffers (New England Biolabs) at a final concentration of 1% NP40 and 50mM sodium phosphate before the addition of 100 units of PNGase F andincubated at 37° C. for 4 hours.

FIG. 17A illustrates a shift in the migration of Pro104 protein fromboth the HeLa cell line and ovarian cancer samples when treated withPangs. These results demonstrate not only that Pro104 is glycosylated,but that anti-Pro104 antibodies are capable of detecting bothglycosylated and deglycosylated forms of native Pro104.

Pro104 GPI-Linkage Characterization by PI-PLC

HeLa cells were seeded in 6 well plates. 48 hours later, at 90%confluence, the media were replaced with 1 ml fresh growth media, withand without 0.5 unit phosphatidylinositol-specific phospholipase C(PI-PLC, Sigma). After one hour incubation at 37° C., the media wereharvested and briefly microfuged. 15 μl of unconcentrated media wereanalyzed by SDS-PAGE. Cells were solubilized for immunoblot analysis asdescribed above.

Since human Pro104 was predicted to be a GPI-linked protein, this wastested by treating live HeLa cells with phosphatidylinositol-specificphospholipase C (PI-PLC) as described above. PI-PLC cleaves the membraneanchor from GPI-linked proteins and releases the protein into themedium. FIG. 17B demonstrated no Pro104 protein was shed into the mediumof untreated HeLa cells, however, treatment with PI-PLC released Pro104into the medium where it could be detected by immunoblot The PI-PLCtreatment did not release other non-GPI-linked membrane proteinsindicating that the release of Pro104 was due to specific cleavage ofthe GPI-anchor by the PI-PLC. This experiment shows that Pro104 islocalized to the surface of tumor cells via a GPI-linkage.

Pro104 Biotinylation

Attached Cells

Caco2, CaOV3, or HeLa cells were removed from a 37° C. incubator, placeon ice, and remained on ice for duration of the experiment. Cells werewashed 3 times with ice cold PBS (10 mM Na—P) at pH 7.4. Biotinylationreagent (Sulfo-NHS-SS-Biotin; Pierce, Rockford, Ill.) dissolved in icecold PBS to final concentration of 0.5 mg/ml was added to cover thecells completely (approximately 200 μl) and incubated on ice for 30minutes. Biotinylation reagent was removed and cells were washed with1×PBS+25 mM Tris once followed by three washes with ice cold PBS. 500 μlof Lysis Buffer (1×PBS+1.0% Triton) with 1× protease inhibitors wasadded to cells and incubated on ice for 10 minutes. Resulting lysate wastransferred into a microcentrifuge tube and spun for 2 minutes at 14,000rpm at 4° C. 50 μl of supernatant was saved to be run on gels as totalprotein extract, while the remaining volume of supernatant wasimmunoprecipitated with 20 μl of Streptavidin Agarose beads (Pierce).After immunoprecipitation, the beads were washed three times with celllysis buffer (1×PBS+1.0% Triton). 100 μl of 1×LDS Sample Buffer (NuPage;Invitrogen) and 1× Sample Reducing Agent (NuPage; Invitrogen) were addedto each sample and incubated at 70° C. for 10 minutes prior to runningon gel. A standard western blot was then performed as described above.

Detached Cells

Caco2, CaOV3, or HeLa cells were removed from a 37° C. incubator, placeon ice, and remained on ice for duration of the experiment Cells weredetached and washed 3 times with ice cold PBS (10 mM Na—P) at pH 7.4 andresuspended in 500P4 PBS. Biotinylation reagent (Sulfo-NHS-SS-Biotin;Pierce, Rockford, Ill.) dissolved in ice cold PBS with a concentrationof 1.0 mg/ml was added to the cells for a final concentration of 0.5mg/ml and incubated on ice for 30 minutes. Cells were spun at 200 rpmfor 5 minutes. Biotinylation reagent was removed and cells were washedwith 1×PBS+25 mM Tris once followed by three washes with ice cold PBS.Cells were spun at 200 rpm for 5 minutes in between washings to removebuffer. 500 μl of Lysis Buffer (1×PBS+1.0% Triton) with 1× proteaseinhibitors was added to cells and incubated on ice for 10 minutes.Resulting lysate was transferred into a microcentrifuge tube and spunfor 2 minutes at 14,000 rpm at 4° C. Supernatant was transferred to anew microcentrifuge tube and protein concentration was determined usingthe BCA assay (Pierce). 50 μl of supernatant was saved to be run on gelsas total protein extract, while the remaining volume of supernatant wasimmunoprecipitated with 20 μl of Streptavidin Agarose beads (Pierce).After immunoprecipitation, the beads were washed three times with celllysis buffer (1×PBS+1.0% Triton). 100 μl of 1×LDS Sample Buffer (NuPage;Invitrogen) and 1× Sample Reducing Agent (NuPage; Invitrogen) were addedto each sample and incubated at 70° C. for 10 minutes prior to runningon gel. A standard western blot was then performed as described above.

FIG. 18 demonstrates that native Pro104 is biotinylated on the cellsurface compared to NaK-ATPase (positive control) and GAPDH (negativecontrol).

The fact that Pro104 is located on the cell surface via GPI-Linkage incell lines makes it an ideal target for antibody based therapy.Furthermore, binding of anti-Pro104 antibodies to glycosylated anddeglycosylated Pro104 on cell lines and ovarian cancer cells indicatesanti-Pro104 antibodies are suitable for immunotherapy of tumors with orwithout conjugated drugs, toxins, enzymes, prodrug activating moleculesor isotopes.

Example 6 Generation of Pro104 Expressing Cell Lines

Cells and Cell Cultures

SKOV3, RK3E, 293T, HeLa, CaOV3, NCIH522 and HCT116 cell lines werepurchased from American Type Culture Collection (Manassas, Va.). Cellswere grown in DMEM (Invitrogen Life Technologies, Carlsbad, Calif.) withL-glutamine plus 4.5 g/L glucose and supplemented with 10% FBS and 100U/mL Penicillin/Streptomycin (Cellgro, Herndon, Va.). All cells weremaintained in a humidified 37° C. incubator with 5% CO₂.

Expression Vector Construction

As a source for cloning of Pro104, human ovarian cancer cDNA wasprepared from poly-A+ mRNA using a BD SMART PCR cDNA synthesis kit (BDBioscience/Clontech, Palo Alto, Calif.). For construction of aretroviral expression vector encoding untagged Pro104 (pLXSN-Pro104),Pro104 cDNA was synthesized by PCR reaction using ovarian cancer5′-RACE-ready cDNA as template and the following gene specific primers:5′-end: 5′-ATGGGCGCGCGCGGGGCGCTGCTG (SEQ ID NO: 8) CTG-3′ 3′-end:5′-TTATCAGACCGGCCCCAGGAGTGG (SEQ ID NO: 9) GAGAGCCCA-3′The PCR fragment was cloned into the Hpa I cloning site of the pLXSNvector (BD Bioscience/Clontech) and sequence verified. The Genbankaccession for pLXSN vector is #M28248.

For construction of a retroviral expression vector encoding Pro104 withan in-frame COOH-terminal hemagglutinin tag (pLXSN-Pro104HA), the sameprocedure was used except that the 3′ primer used was: 3′-end (HA tag isbold): 5′-TTATCACGCGTAGTCCGGCACGTCGTACGGG (SEQ ID NO: 10) TAGCCGACCGGCCCCAGGAGTGGGAGAGCCCA- 3′

The pLXSN retrovirus vector utilizes a 5′Mo-MuSV (Moloney Murine SarcomaVirus) LTR and 3′Mo-MuLV (Moloney Murine Leukemia Virus) LTR to driveexpression of cDNA's cloned into the multiple cloning site and an SV40promoter driving expression of a Neo^(r) gene encoding G418 resistance.pLAPSN, a retroviral expression vector encoding alkaline phosphatase(AP), was purchased from BD Bioscience/Clontech (referred to aspLXSN-AP).

Virus Production

Ecotropic virus was used to infect RK3E cells and amphotropic virus toinfect SKOV3 cells. For ecotropic virus packaging, one day prior totransfection, 293T cells were seeded at a density of 8×10⁵ cells perwell of a 6 well dish onto Biocoat collagen coated plates (BD). Cellswere transfected with purified plasmid DNA's using Lipofectamine withthe addition of PLUS reagent (Invitrogen Life Technologies, Carlsbad,Calif.). Per well of cells 0.8 μg of virus plasmid DNA: pLXSN-Pro104,pLXSN-Pro104HA or pLXSN-AP plus 0.8 μg pVpack-ECO and 0.8 μg pVpackGP(Stratagene, La Jolla, Calif.) were added to a stock of 125 μL DMEMwithout serum and 10 μL of PLUS reagent followed by incubation for 15minutes at room temperature. Subsequently, 8 μL of lipofectamine dilutedinto 125 μL of DMEM medium were added to the DNA/PLUS reagent mixtureand incubated for 15 minutes as room temperature. One ml of DMEM wasadded to the final Lipofectamine/DNA mixture and applied to the cellmonolayer, already containing 1 ml DMEM without serum, followed byincubation at 37° C. for 3 hours. One mL of DMEM containing 20% FBS wasadded to the transfection mix after the 3 h incubation and grownovernight. Finally, the media was changed to DMEM supplemented with 10%FBS+100 U/mL Pen/Strep for virus collection. Virus-containing media wereharvested 24 hours later and filtered through a 0.45 μm polysulfonicfilter.

For amphotropic virus packaging the same procedure was followed exceptthat the pVpack Ampho plasmid (Stratagene) was used instead of thepVpack Eco plasmid.

Virus Infection and Selection

Polybrene (Hexadimethrine Bromide; Sigma, Saint Louis, Mo.) was added tofresh virus-containing medium at a final concentration of 4 μg/ml. RK3Eor SKOV3 cells, plated the day before at a density of 3×10⁵ cells per100 mm² dish, were washed once with phosphate-buffered saline includingCa2+ and Mg2+ (Cellgro). The virus solution (6 ml per 100 mm2 dish) wasapplied directly to the cells and then incubated for 3 hours in ahumidified 37° C. incubator with 5% CO₂ with occasional swirling. Thevirus-containing medium was replaced by fresh growth medium and thecells incubated at 37° C. for 60-72 hours at which point a finalconcentration of 350 ug/mL of G418 sulfate (Cellgro) was included in thegrowth medium to select for virus-infected cells. Cells were maintainedbetween 70-80% confluence and G418-containing media was changed every 2days. Following G418 selection, pools of cells were used for subsequentexperiments including verification of Pro104 protein expression byWestern immunoblot analysis where cells were extracted and analyzed asdescribed above. Expression of AP by infected cell monolayers wasmonitored by staining whereby monolayers of cells were fixed for 10minutes at room temperature with a solution of 0.5% glutaraldehyde,rinsed with PBS, heated to 65° C. for 30 minutes and AP visualized byincubation with BCIP/NBT liquid substrate (Sigma, Saint Louis, Mo.) for2-3 hours.

Results of Cell Line Virus Infection and Selection

SKOV3, RK3E, 293T, HeLa, CaOV3, NCIH522 and HCT116 cell lines underwentvirus infection and selection to overexpress Pro104.

Retroviral-mediated overexpression of Pro104 protein in RK3E cells wasconfirmed by Western Immunoblot. FIG. 19 is a western immunoblotdemonstrating Pro104 protein expression in retroviral packaging celllines and virus infected RK3E cells.

Retroviral-mediated overexpression of Pro104 protein in SKOV3 cells wasconfirmed by Western Immunoblot FIG. 20 is a western immunoblotdemonstrating Pro104 protein expression in retroviral packaging celllines and virus infected SKOV3 cells.

Example 7 siRNA Generation and Transfection

siRNA Oligonucleotide Design and Preparation

To design Pro104 specific siRNA molecules, sequences were selected fromthe open reading flame of the Pro104 mRNA based on methods previouslydescribed (Elbashir et al., 2001, Nature 411:494-498A random “scrambled”siRNA sequence which should not generate knockdown of any known cellularmRNA was used as a negative control. As an additional negative control asiRNA targeting Emerin was used to demonstrate that knockdown of anon-essential mRNA did not affect Pro104 levels nor any of thebiological endpoints studied. As a positive control for knockdown of anmRNA leading to apoptosis induction, a siRNA targeting either DAXX orOPAI was used, based on published data. Michaelson et al., 2002, Journalof Cell Science, 116:345-352; Olichen et al., 2003, J Biol Chem.,278(10):7743-6. A BLAST search against the human genome was performedwith each selected siRNA sequence to ensure that the siRNA wastarget-specific and would not function to knockdown other sequences. ThesiRNA sequences used to knockdown Emerin and DAXX were obtained frompublished papers. Michaelson et al., 2002; Harborth et al., 2001,Journal of Cell Science, 114:4557-4565.

All siRNA molecules (HPP purified grade) were chemically the synthesizedby Xeragon Inc. (Germantown, Md.). siRNA's were dissolved in sterilebuffer, heated at 90° C. for 1 minute and then incubated at 37° C. for 1hour prior to use. siRNA oligonucleotides with two thymidine residues(dTdT) at the 3′ end of the sequence consisted of the following specificRNA sequences: Pro104 #56: sense 5′-CACAUCCAGCCCAUCUGUC-3′ (SEQ ID NO:11) Pro104 #79: sense 5′-GAGGAUGAGGCACUGCCAU-3′ (SEQ ID NO: 12) Pro104#80: sense 5′-CUCUAUGUGCAACCACCUC-3′ (SEQ ID NO: 13) Pro104 #81: sense5′-GUACAGUUUCCGCAAGGAC-3′ (SEQ ID NO: 14) Scrambled: sense5′-UUCUCCGAACGUGUCACGU-3′ (SEQ ID NO: 15) Emerin: sense5′-CCGUGCUCCUGGGGCUGGG-3′ (SEQ ID NO: 16) DAXX: sense5′-GGAGUUGGAUCUCUCAGAA-3′ (SEQ ID NO: 17) OPAI sense5′-GUUAUCAGUCUGAGCCAGG-3′ (SEQ ID NO: 18)

Additional siRNA oligonucleotides specific for Pro104 with two thymidineresidues (dTdT) at the 3′ end of the sequence consisted of the followingspecific RNA sequences: Pro104_siRNA#1: gccggagucgcaggaggcg (SEQ ID NO:19) Pro104_siRNA#2: cucgggcguuggccguggc (SEQ ID NO: 20) Pro104_siRNA#3:accuauagugaccuuagug (SEQ ID NO: 21) Pro104_siRNA#4: ccuauagugaccuuaguga(SEQ ID NO: 22) Pro104_siRNA#5: uucacccuaugacauugcc (SEQ ID NO: 23)Pro104_siRNA#6: gcugucugcaccugucacc (SEQ ID NO: 24) Pro104_siRNA#7:ccggacagacugcugggug (SEQ ID NO: 25) Pro104_siRNA#8: agaggaugaggcacugcca(SEQ ID NO: 26) Pro104_siRNA#9: guucaggucgccaucauaa (SEQ ID NO: 27)Pro104_siRNA#10: ggacaucuuuggagacaug (SEQ ID NO: 28) Pro104_siRNA#11:caagaauggacugugguau (SEQ ID NO: 29) Pro104_siRNA#12: gaauggacugugguaucag(SEQ ID NO: 30) Pro104_siRNA#13: uggacugugguaucagauu (SEQ ID NO: 31)Pro104_siRNA#14: ucggcccggugucuacacc (SEQ ID NO: 32) Pro104_siRNA#15:uaucagccaccacuuugag (SEQ ID NO: 33) Pro104_siRNA#16: gucaggcccugguucucuu(SEQ ID NO: 34) Pro104_siRNA#17: uaaacacauuccaguugau (SEQ ID NO: 35)Pro104_siRNA#18: uaaacacauuccaguugau (SEQ ID NO: 36) Pro104_siRNA#19:acacauuccaguugaugcc (SEQ ID NO: 37) Pro104_siRNA#20: cacauuccaguugaugccu(SEQ ID NO: 38)Transfection with siRNA Oligonucleotides

HeLa (4×10⁴ cells) and CaOV3 (6×10⁴ cells) cells expressing Pro104 wereseeded in 12-well plates for 18-24 hours prior to transfection.Transient transfection was carried out using Oligofectamine reagentInvitrogen Life Technologies, Carlsbad, Calif.) according to themanufacturer's protocol. A final concentration of 100 nM siRNA (exceptDAXX siRNA which was 200 nM) and 1.5 ul Oligofectamine were used perwell of cells. Pro104, Scrambled, DAXX and Emerin siRNA's weretransfected in triplicate for all experiments. Parallel wells of cellswere evaluated 72 hours after transfection for changes in mRNA levels byquantitative real-time RT-PCR (QPCR), changes in protein levels byWestern immunoblot and changes in apoptosis by two different assaysystems (see below). All findings were confirmed with at least 2additional experiments.

Example 8 SDS-PAGE and Western Immunoblot Analysis

72 hrs after transfection with siRNA, cell extracts were prepared on iceusing modified RIPA buffer (1% NP40, 10 mM Na₂PO₄, 0.15M NaCl) plus aprotease inhibitor cocktail (Roche Inc.). Between 20 and 50 ug ofprotein extract were used for each gel lane; protein equivalentconcentrations were evaluated for protein level comparisons on the samegel. Clarified extracts were mixed with an equal volume of 2×concentrated Laemmli sample buffer Invitrogen Life Technologies,Carlsbad, Calif.), heated to 70° C. for 10 minutes and then analyzedusing pre-cast 4-12% SDS-polyacrylamide minigels (Nupage; InvitrogenLife Technologies, Carlsbad, Calif.) with MES running buffer (Nupage;Invitrogen Life Technologies, Carlsbad, Calif.). Gels were transferredto Immobilon-P PVDF membranes with a 0.45 μm pore size (Invitrogen LifeTechnologies, Carlsbad, Calif.) using 1× Nupage transfer buffer plus 10%Methanol. The membranes were rinsed and blocked for 1 hour at roomtemperature using 5% nonfat dry milk in PBS with 0.05% Tween-20.Membranes were incubated with primary antibody overnight in 5% nonfatdry milk in PBS with 0.05% Tween-20. A mouse monoclonal antibodydirected against Pro104 was produced in house using recombinantbacterial Pro104 protein. The Pro104 monoclonal antibody was diluted1:1000 for a final concentration of 1 ug/ml and a mouse monoclonalantibody against GAPDH (Chemicon Inc., Temecula, Calif.) was diluted1:5000 (for a final concentration of 0.2 ug/ml). Following primaryantibody incubation, membranes were washed four times at roomtemperature for 10 min. each in 1×PBS with 0.05% Tween-20. Horseradishperoxidase linked goat anti-mouse immunoglobulin (Jackson Lab Inc., BarHarbor, Me.) was used (1:10,000 dilution) in 5% nonfat dry milk in PBSplus 0.05% Tween-20 for 1 hour at room temperature to detect the primarymonoclonal antibody. Membranes were finally washed four times for 10min. in 1×PBS plus 0.05% Tween-20 followed by detection using enhancedchemiluminescence (ECL) reagent per manufacturer's directions (Amersham,Piscataway, N.J.) and exposure to X-ray film (Kodak, Rochester, N.Y.).For the Western immunoblot experiment comparing RK3E cells infected withan AP(alkaline phosphatase)-expressing retrovirus with the same cellsinfected with a Pro104-expressing retrovirus, cells were plated ingrowth medium containing either 1% or 10% FBS for 48 hours. Cellextracts were prepared using modified RIPA buffer including aphosphatase inhibitor cocktail (Calbiochem) and 25 ug of clarifiedextract were evaluated by SDS-PAGE and Western immunoblot with apolyclonal antibody specific for the phosphorylated EGF receptor(BioSource International, Camarillo, Calif.).

Effects of Pro104 specific siRNA on Pro104 protein was determined bywestern immunoblot FIG. 21 shows siRNA Mediates Specific Down-Regulationof Pro104 Protein in HeLa Cells. A 60% knockdown (ACT=1.3) of Pro104protein was observed. These results were not confined to a single celltype. FIG. 22 shows siRNA Mediates Specific Down-Regulation of Pro104Protein in CaOV3 Cells. A 55% knockdown (ACT=1.2) of Pro104 protein wasobserved.

Example 9 Quantitative Real Time RT-PCR (QPCR)

A QuantiTech SYBR Green RT-PCR kit from Qiagen Inc. was used for QPCRevaluation. The final reaction volume was 20 ul including 10 ul RT-PCRMaster Mix, 2 ul forward primer (5 uM), 2 ul reverse primer (5 uM),QuantiTect RT mix 0.2 ul and RNase-free water. Between 20 and 40 ng oftemplate RNA was used per reaction. QPCR was performed using a Taqman7700 Sequence Detection system (Applied Biosystem Inc.) with thefollowing cycle conditions: 50° C. for 30 min., 95° C. for 15 min., 40cycles at 94° C. for 15 s, 55° C. for 30 s, 72° C. for 30 s, then heldat 72° C. for 2 min.

The QPCR assay was used to determine the effect of Pro104 siRNA on genetranscription levels.

QPCR assays demonstrated Pro104 siRNA specifically knockdown Pro104 mRNAin CaOV3 cells. FIG. 23A shows that Pro104 siRNA do not knockdownnon-Pro104 mRNA (GAPDH) compared to negative controls in CaOV3 cells.FIG. 23B demonstrates Pro104 siRNA knockdown Pro104 mRNA compared tonegative controls in CaOV3 cells.

Specificity of Pro104 siRNA was not limited by cell type. QPCR assaysfurther demonstrated Pro104 siRNA specifically knockdown Pro104 mRNA inHeLa cells. FIG. 24A shows that Pro104 siRNA do not knockdown non-Pro104mRNA (GAPDH) compared to negative controls in HeLa cells. FIG. 24Bdemonstrates Pro104 siRNA knockdown Pro104 mRNA compared to negativecontrols in HeLa cells. A 75% knockdown (ACT-2) of Pro104 was observedin HeLa cells.

Knockdown of essential mRNA such as DAXX leads to apoptosis induction.Michaelson 2002 supra; Olichen 2002, supra. QPCR experimentsdemonstrated that knockdown of Pro104 mRNA may lead to apoptosisinduction as well. FIG. 25 shows Pro104 siRNA knockdown of Pro104 mRNAin HeLa cells compared to a positive control for apoptosis induction(DAXX).

Furthermore, QPCR assays confirmed that different Pro104 siRNA knockdownPro104 mRNA in HeLa cells. FIG. 26B demonstrates that Pro104 siRNAs #56(SEQ ID NO: 10), #79 (SEQ ID NO: 11), #80 (SEQ ID NO: 12) and #81 (SEQID NO: 13) knockdown Pro104 mRNA compared to the negative control(scrambled siRNA). Knockdown of Pro104 mRNA by four different siRNAspecifically designed for Pro104 mRNA is indicative that siRNA designedto interfere with Pro104 mRNA may knockdown Pro104 mRNA and proteinexpression.

Example 10 Apoptosis Assays

Two different assay kits, Annexin V assay and Caspase assay, were usedto evaluate the effects of siRNA on apoptosis.

With the “Apo-ONE Homogeneous Caspase-3/7 Assay” kit (Promega Inc.,Madison, Wis.) the test cells were solubilized directly in the cultureplate and caspase activity, reflected as a fluorescent readout, wasmeasured according to supplier's instructions.

With the second kit, “Guava Nexin V-PE Kit” (Guava Technologies Inc.),treated cells were harvested by trypsinization and washing andapproximately 10⁵ cells were resuspended in 40 ul provided buffer and 5ul each Annexin V (+) and 7-AAD(−) were added. Following 20 minutesincubation on ice, cells were analyzed using the Guava PCA machineaccording to manufacturer's instructions.

Annexin V assay results demonstrates that different Pro104 siRNA whichknockdown Pro104 mRNA induces apoptosis. FIG. 26A shows that Pro104siRNAs #56 (SEQ ID NO: 10), #79 (SEQ ID NO: 11), #80 (SEQ ID NO: 12) and#81 (SEQ ID NO: 13) or DAXX siRNA induce apoptosis compared to scrambledsiRNA (negative control) in HeLa cells.

Annexin V assay results also demonstrate specific knockdown of Pro104mRNA with Pro104 siRNA induces cell death. FIG. 27A shows a greaterpercentage of HeLa cells are early apoptotic when transfected withPro104 siRNA compared to negative controls (no siRNA, and scrambledsiRNA). Additionally, FIG. 27B shows a greater percentage of HeLa cellsare necrotic when transfected with Pro104 siRNA compared to negativecontrols (no siRNA, and scrambled siRNA).

Induction of apoptosis by knockdown of Pro104 mRNA by Pro104 siRNA wasdemonstrated by the Anexin V assay, and the Caspase assay. Results ofAnnexin V assay in FIG. 28A show a greater percentage HeLa cells areapoptotic when transfected with Pro104 siRNA, or DAXX siRNA (positivecontrol) compared to scrambled siRNA (negative control). Results ofCaspase assay in FIG. 28B show a greater percentage HeLa cells areapoptotic when transfected with Pro104 siRNA, or DAXX siRNA (positivecontrol) compared to scrambled siRNA (negative control).

Induction of apoptosis by Pro104 mRNA knockdown by Pro104 siRNA was notlimited by cell type. Results of Annexin V assay in FIG. 29 show agreater percentage CaOV3 cells also are apoptotic when transfected withPro104 siRNA, or OPAI siRNA (positive control) compared to scrambledsiRNA (negative control) and no siRNA (negative control).

Induction of apoptosis by knockdown of Pro104 mRNA is due loss of Pro104function. Pro104 siRNA does not induce apoptosis in cells that do notexpress Pro104. FIG. 30A demonstrates knockdown levels of Pro104 mRNA,Emerin mRNA-(positive control, non-essential) and DAXX mRNA (positivecontrol, essential) in SKBR3 cells which do not express Pro104 mRNA.There is no difference between knockdown levels of Pro104 mRNA due toscrambled siRNA and Pro104 specific siRNA while Emerin and DAXX mRNAlevels are knocked-down, 50% and 65% respectively, by specific siRNAcompared to scrambled siRNA.

Results from a Caspase assay in FIG. 30B demonstrate that SKBR3 cellswhich do not express Pro104 mRNA do not undergo apoptosis whentransfected with Pro104 siRNA while apoptosis is induced by transfectionby DAXX siRNA (positive control). Furthermore, SKBR3 cells which do notexpress Pro104 mRNA do not undergo apoptosis when transfected withEmerin siRNA (non-essential) or scrambled siRNA (negative control).

Results from FIGS. 30A and 30B serve as a negative control to showPro104 siRNA transfection induces apoptosis by specifically knockdown ofPro104 mRNA and down-regulation of Pro104 protein.

Furthermore, Pro104 is shown to be essential to cell survival. FIGS. 30Aand 30B demonstrate that knockdown of non-essential mRNA (Emerin) doesnot induce apoptosis compared to scrambled siRNA (negative control).Only knockdown of essential mRNA such as DAXX (positive control) andPro104 will induce apoptosis.

Example 11 Soft Agar Assay

Soft agar assays were conducted using 6-well plates (Corning, VWR). The2 ml bottom agar base layer consisted of 0.8% agar, 10% FBS in Iscove'smedium (Invitrogen Life Technologies, Carlsbad, Calif.). Trypsinizedcells were suspended in 0.4% agar, 10% FBS in Iscove's medium andapplied in a 5 ml final volume on top of the solidified base layer.Three different viable cell numbers, 10⁵, 10⁴ and 5×10³ cells, wereseeded in agar per 6 cm² well in duplicate. A final 2 ml layerconsisting of 0.8% agar, 10% FBS in Iscove's medium was applied on topof the solidified cell layer. The agar plates were then incubated in ahumidified 37° C. incubator with 5% CO₂ for approximately 2 weeks beforecolonies appeared. The soft agar was maintained by weekly feedings withgrowth medium. Colonies were counted between 2 and 4 weeks. 24 to 36hours after siRNA transfection, HeLa cells were trypsinized and platedin soft agar at a density of 10⁴ cells per well as described above.

Soft agar assays were conducted to evaluate the effects ofover-expression of Pro104, Pro104 protease activity and knockdown ofPro104 on cells.

FIG. 31 demonstrates that over expression of Pro104 induces cell growthin soft agar. Table 8 below shows the number of colonies observed insoft agar plates for each cell type in FIG. 31. TABLE 8 Number ofColonies in Soft Agar Plates Number of FIG. Cell Type Colonies 31ARK3E-AP 0 31B RK3E-Pro104 60 31C RK3E-Pro104-HA 68 31D NCIH522(−control) 0 31E HCT116 (+control) >200Pro104 Protease Activity is Required for Cell Growth

RK3E cells were infected with retrovirus vectors expressing wild-typePro104 protein (Pro104), with and without a C-terminal hemagglutinin tag(IA). Additionally, RK3E cells were infected with retrovirus vectorsexpressing Pro104 protein lacking enzymatic activity with a pointmutation within the catalytic triad (Pro104-mut) or Alkaline Phosphatase(AP-control). Retroviral infection was followed by G418 selection forinfected cells.

Expression of Pro104 proteins in the G418-selected cell pools wasverified by immunoblot with a monoclonal antibody directed againstPro104, FIG. 32A. Expression of AP in the G418-selected cells wasevaluated by staining cell monolayers for AP activity which showed thatessentially all of the cells were positive (FIG. 32B) and, therefore,most of the G418-selected cells were expressing the gene of interest.The virus-infected, selected cells were then plated in soft agar andmonitored for colony formation. The parental RK3E cells did not form anycolonies under the conditions used for the assay nor did theAP-expressing cells (FIGS. 32C, 32D). However, cells expressing eitherHA-tagged (Pro104-HA) or untagged Pro104 protein formed coloniesdemonstrating that ectopic expression of the protein can promotetransformation (FIGS. 32C, 32D). The mutant Pro104 (Pro104-mut) proteinwas unable to induce soft agar growth of RK3E cells (FIGS. 32C, 32D)indicating that the catalytic function of Pro104 is required fortransformation.

Knockdown of Pro104 mRNA by siRNA Inhibits Cell Growth

As shown above, specific knockdown of Pro104 mRNA and protein in Helacells led to an increase in apoptosis, measured by two differentmethods. We next examined whether knockdown of Pro104 could affect theability of HeLa cells to form colonies in soft agar. HeLa cells weretreated with scrambled, Pro104- or DAXX-specific siRNA and subsequentlyplated in soft agar to evaluate colony formation. Scrambled siRNA servedas a negative control while DAXX-specific siRNA served as positivecontrol for inducing apoptosis. HeLa cells form numerous large coloniesin agar and this was not affected by the scrambled siRNA as demonstratedin FIGS. 33C and 33F. In contrast, both Pro104- and DAXX-specificsiRNA's inhibited the number of colonies formed by approximately 88% and80%, respectively (FIGS. 33A and 33B, respectively). Furthermore, thesize and morphology of colonies formed by cells treated with Pro104 andDAXX-specific siRNAs was smaller and restricted (FIGS. 33D and 33E,respectively).

QPCR performed after transfection with siRNA showed that the Pro104,DAXX and Emerin mRNA levels were decreased compared to transfection withscrambled siRNA (FIG. 34A). In this experiment the Pro104 and DAXXsiRNA's were again able to induce caspase activity whereas the scrambledand Emerin-specific siRNA did not (FIG. 34B). A siRNA against emerin hadno effect on the ability of the Hela cells to form colonies.

Results

These assays confirm that Pro104 is essential to cell survival andover-expression induces cell growth (FIG. 31). Knockdown of Pro104 mRNAby siRNA (FIGS. 23-26) reduces protein expression (FIGS. 21, 22) whichinduces apoptosis (FIGS. 27-30). This in turn results in fewer coloniesand colony size and morphology indicative of apoptosis in soft agar(FIG. 33). Mutated Pro104 lacking protease activity does not induce cellgrowth confirming Pro104 activity is essential for cell growth (FIG.32).

Example 12 Tumor Xenograft Experiment

To further evaluate the transforming ability of Pro104, RK3E cellsexpressing Pro104 or AP were implanted subcutaneously into Nude orSCID/Beige mice and tumor formation was monitored.

Increased Growth of Ovarian Tumor Cells Over-Expressing Pro104 in NudeMice

Retrovirus-infected, G418-selected pools of SKOV3 or RK3E cellsexpressing either AP or Pro104 were injected subcutaneously into nudemice. Parental SKOV3 cells were also used for comparison. For SKOV3cells, 10⁷ of each type were implanted with matrigel into each of 6mice. For RK3E cells, 5×10⁶ cells were implanted into each of 8 micewithout matrigel. Tumor formation was monitored by palpation and calipermeasurement where possible every 4 days for a period of 4 weeks.

An animal model demonstrated growth of human ovarian tumor cellsover-expressing Pro104. SKOV3 cells over-expressing Pro104 increased involume compared to Parental SKOV3 cells (control) or AP expressing SKOV3cells (non-growth inducing control).

Additionally, table 9 below shows over-expression of Pro104 promotestumor formation in subcutaneous cell xenografts in nude mice. TABLE 9Tumor formation in subcutaneous cell xenografts. RK3E Cell Line # micewith nodules* or 5 × 10⁶ cells implanted tumor** by 4 weeks AP (negativecontrol) 0/8 Pro104  8/8* V-Ras (positive control)  8/8**

These animal models demonstrate that Pro104 overexpressing cells growand form nodules.

Increased Growth of Ovarian Tumor Cells Over-Expressing Pro104 in SCIDMice

Retrovirus-infected, G418-selected pools of SKOV3 or RK3E cellsexpressing either AP or Pro104 were injected subcutaneously intoSCID/Beige mice (Charles River Laboratories). Nine or ten mice were usedper group as indicated. For SKOV3 cells, 10⁷ cells in 100 ul PBS wereimplanted with matrigel and for RK3E cells, 5×10⁶ cells in 100 μl PBSwere implanted without using matrigel. Tumor formation was monitored bypalpation and caliper measurement and tumor volume was calculated usingthe formula: (length×width²)/2. The graphs shown in FIGS. 36A and 36Cplot mean group tumor volume over time. All animal experiments wereperformed in complete compliance with institutional guidelines.

For the SKOV3 xenograft studies a single factor ANOVA was performed totest whether on the last day of measurement the tumor volumes betweencontrol and Pro104 groups differed. The results indicated a >99.0%probability that the two groups do not have the same tumor volume.Furthermore, Pairwise Two-Sample t-Tests Assuming Unequal Variances withBonferroni Correction analysis were performed comparing theSKOV3-testisin tumors to the SKOV3-control tumors. Analysis of data fromthe last day of measurement revealed that the SKOV3-Pro104 tumors hadsignificantly larger volumes than SKOV3-control tumors at a 99.0%confidence level.

KR3E-Pro104 Tumor Cell Growth

Nine out of nine mice implanted with Pro104 expressing RK3E cellsdeveloped large tumors whereas none of the mice implanted withAP-expressing cells formed tumors (FIG. 35A). At the conclusion of thexenograft study tumors were harvested and evaluated by immunoblot forthe presence of Pro104 protein. The tumors maintained expression ofPro104 protein at a level similar to that observed in the infected RK3Ecells prior to implantation (FIG. 35B).

SKOV3-Pro104 Tumor Cell Growth

We next evaluated the effect of ectopic Pro104 expression on tumorformation by the human SKOV3 ovarian cancer cell line which was chosenfor this purpose since it does not express endogenous Pro104 mRNA norPro104 protein (evaluated by immunoblot-FIG. 35D). SKOV3 cells wereinfected with either a retrovirus expressing Pro104 or the AP controlfollowed by G418-selection. Expression of Pro104 protein in the selectedcells was verified by immunoblot (FIG. 35D). AP control andPro104-expressing SKOV3 cells were implanted subcutaneously intoSCID/Beige mice and monitored for tumor formation. SKOV3 cancer cellsare known to form tumors as xenografts in mice. As expected, the APcontrol-expressing SKOV3 cells were also capable of growth as xenograftswhere 10 out of 10 mice implanted formed tumors (FIG. 35C). However,cells expressing ectopic Pro104 protein formed larger tumors throughoutthe time course when compared to the AP-control cells (FIG. 35C).Statistical analysis of the data showed that the increased size ofSKOV3-Pro104 tumors compared to AP control-SKOV3 tumors was significant.

Example 13 Anti-Pro104 Molecules in Combination with Anti-Angiogenesisand Anti-Vascular Molecules

Angiogenesis plays a critical role in many physiological processes, suchas embryogenesis, wound healing, and menstruation and in certainpathological events, such as solid tumor growth and metastasis,arthritis, psoriasis, and diabetic retinopathy as described above.

Additionally, vascular targeting agents, which selectively destroy tumorblood vessels, may be attractive agents for the treatment of solidtumors. They differ from anti-angiogenic agents in that they target themature, blood-conducting vessels of the tumors. They are better suitedfor larger tumors where angiogenesis can occur less frequently. Vasculartargeting agents include antibodies which bind to specific targets orcomplexes. For application in man, target molecules are needed that areselectively expressed on the vascular endothelium of tumors.

In addition to targeting Pro104 to modulate growth of Pro104 expressingtumors, targeting of angiogenesis associated molecules or vascularassociated molecules and complexes may be used to enhance anti-Pro104therapies. Specifically, anti-Pro104 antibodies may be used incombination with antibodies which specifically target angiogenesisassociated molecules or vascular associated molecules and complexes toslow, stop, regress, reverse or inhibit growth or metastasis of Pro 104expressing tumors.

See Feng D., et al. J Histochem Cytochem. 2000 April; 48(4):545-56;Brekken R A., et al. Cancer Res. 2000 Sep. 15; 60(18):5117-24; Brekken RA., et al., Anticancer Res. 2001 November-December; 21(6B):4221-9; andBrekken R A., et al., Int J Cancer. 2002 Jul. 10; 100(2):123-30.

Anti-Pro104 Antibodies in Combination with Anti-VEGF Antibodies

Vascular permeability factor/vascular endothelial growth factor(VPF/VEGF) is a potent multifunctional cytokine that permeabilizesvascular endothelium to plasma proteins and reprograms endothelial cellgene expression so as to induce angiogenesis. VPF/VEGF is secreted bymany tumors and by activated macrophages, keratinocytes, synovial cells,various embryonic cells, and cultured epithelial and mesenchymal celllines. There are at least five splice variants of VEGF, encodingproteins of 121, 145, 165, 189, and 206 amino acids. The smallerversions having 121, 145, or 165 amino acids are secreted from cells.Secreted VEGF is an obligate dimer of between Mr 38,000 and Mr 46,000 inwhich the monomers are linked by two disulfide bonds. The VEGF dimerbinds to one of two well-characterized receptors, VEGFR1 (FLT-1) andVEGFR2 (KDR/Flk-11), that are selectively expressed on endothelialcells. A recently identified third cell surface protein, neuropilin-1,binds VEGF165 with high affinity. VPF/VEGF induces its biologicaleffects by binding to these receptors which are selectively expressed invascular endothelium.

Anti-Pro104 antibodies may be used in combination with anti-VEGFantibodies to slow, stop, regress, reverse or inhibit growth ormetastasis of Pro104 expressing tumors.

Anti-Pro104 Antibodies in Combination with Anti-VEGF Receptor Antibodies

VEGFR1 and VEGFR2 are members of the type III receptor tyrosine kinasefamily that is characterized by seven extracellular IgG-like repeats, asingle spanning transmembrane domain, and an intracellular splittyrosine kinase domain. Both receptors are strikingly upregulated intumors, wounds, and in certain types of inflammation (e.g., rheumatoidarthritis, psoriasis) in which VPF/VEGF is overexpressed. The complexthat forms between tumor-secreted VPF/VEGF and its receptors has beenrecognized as an attractive potential target for antiangiogenesistherapy. VEGF binds to VEGFR1 and VEGFR2 with high affinities having aKd (dissociation constant) of 15-100 pM and 400-800 pM, respectively.VEGFR2 appears to be the dominant signaling receptor in VEGF-inducedmitogenesis and permeability.

Expression of both VEGFR-1 and VEGFR-2 has been localized by in situhybridization to microvascular endothelium of normal kidneys and totumors, healing wounds, and inflammatory sites. VEGFR-2 has also beenidentified in the blood vessels of human placentas, breast cancers, andgastric carcinomas by light microscopic immunohistochemistry. See

Anti-Pro104 antibodies may be used in combination with antibodiesagainst VEGFR-1, VEGFR-2 or neuropilin-1, to slow, stop, regress,reverse or inhibit growth or metastasis of Pro104 expressing tumors.

Anti-Pro104 Antibodies in Combination with Anti-Vascular TargetingAntibodies

Vascular targeting antibodies specifically bind to vascular associatedmarkers. Such markers include the complexes that are formed whenvascular endothelial growth factor (VEGF) binds to its receptors(VEGFR). VEGF production by tumor cells is induced by oncogenic genemutations and by the hypoxic conditions within the tumor mass. Thereceptors, VEGFR1 (FLT-1) and VEGFR2 (KDR/Flk-1), are upregulated onvascular endothelial cells in tumors by hypoxia and by the increasedlocal concentration of VEGF. Consequently, there is a high concentrationof occupied receptors on tumor vascular endothelium.

Vascular targeting with monoclonal antibodies that bind to VEGF: VEGFRcomplexes and their use as tumor vascular targeting agents are known tothose of skill in the art Antibodies which blocks VEGF from binding toVEGFR2 but not VEGFR1 might have dual activity as an anti-angiogenicagent by inhibiting VEGFR2 activity and as a vascular targeting agentfor selective drug delivery to tumor vessels.

Anti-Pro104 antibodies may be used in combination with antibodiesagainst VEGF:VEGFR complexes to slow, stop, regress, reverse or inhibitgrowth or metastasis of Pro104 expressing tumors. Examples of antibodiesinclude but are not limited to anti-Pro104, Pro104.C1, Pro104.C4,Pro104.C13, Pro104.C17, Pro104.C18, Pro104.C19, Pro104.C24, Pro104.C25,Pro104.C27, Pro104.C34, Pro104.C37, Pro104.C46, Pro104.C48, Pro104.C49,Pro104.C50, Pro104.C53, Pro104.C54, Pro104.C55, Pro104.C57, Pro104.C60,Pro104.C66, Pro104.C75, Pro104.C84, Pro104.D4, Pro104.D6, Pro104.D9,Pro104.D12, Pro104.D14, Pro104.D18, Pro104.D19, Pro104.D20, Pro104.D21,Pro104.D26, Pro104.D29, Pro104.D31, Pro104.D43, Pro104.D47, Pro104.D51,Pro104.D55, Pro104.D56, Pro104.D58, Pro104.D62, Pro104.D63, Pro104.D64,Pro104.D68, Pro104.D69, Pro104.D75, Pro104.D81, Pro104.D85, Pro104.D88,Pro104.D91, Pro104.D94, Pro104.D102, Pro104.D106, Pro104.D111,Pro104.D112, Pro104.D113, Pro104.D114, Pro104.D115, Pro104.D116,Pro104.D117, Pro104.D118, Pro104.D119, Pro104.D120, Pro104.D121,Pro104.D122, Pro104.D123, Pro104.D, Pro104.D124, Pro104.D125,Pro104.D126, Pro104.D127, Pro104.D, Pro104.D128, Pro104.D129,Pro104.D130, Pro104.D131, Pro104.D132, Pro104.D133, Pro104.D134,Pro104.D135, Pro104.D136, Pro104.D137, Pro104.D138, Pro104.D139,Pro104.K14, Pro104.K15, Pro104.K16, Pro104.K47, Pro104.K71, Pro104.K72,Pro104.K74, Pro104.K75, Pro104.K76, Pro104.K78, Pro104.K81, Pro104.K87,Pro104.K88, Pro104.K89, Pro104.K155, Pro104.K156, Pro104.K157,Pro104.K158, Pro104.K159, Pro104.K160, Pro104.K163, Pro104.K164,Pro104.K176, Pro104.K217, Pro104.K226, Pro104.K227, Pro104.K240,Pro104.K274, Pro104.K264, Pro104.K281, Pro104.K358 or Pro104.K362;anti-VEGF, bevacizumab (Avastin; Genentech Inc., South San Francisco,Calif.; Rini et al. Clin Cancer Res. 2004 Apr. 15; 10(8):2584-6),infliximab (Canete et al. Arthritis Rheum. 2004 May; 50(5):1636-41,Klimiuk et al. Arch Immunol Ther Exp (Warsz). 2004 January-February;52(1):3642); anti-VEGF-R, vatalanib (Manley et al. Biochim Biophys Acta.2004 Mar. 11; 1697(1-2): 17-27); anti-VEGF-R2, DC101 (Tong et al. CancerRes. 2004 Jun. 1; 64(11):3731-6, Kiessling et al. Neoplasia. 2004May-June; 6(3):213-23); anti-VEGF-3, hF4-3C5 (Persaud et al. J Cell Sci.2004 Jun. 1; 117(Pt 13):2745-56). In addition, combination therapy forEGFR may be used e.g. anti-EGFR, cetuximab, C225 (Andre et al. BullCancer. 2004 January; 91(1):75-80), gefitinib, ZD1839(Ciardiello et al.Clin Cancer Res. 2004 Jan. 15; 10(2):784-93).

Example 14 Deposit of Cell Lines and DNA

Hybridoma cell lines were deposited with the American Type CultureCollection (ATCC) located at 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and accorded accession numbers.

The following hybridoma cell lines were deposited with ATCC,Pro104.C55.1, Pro104.C25.1, Pro104.D9.1 and Pro104.K81.15. The names ofthe deposited hybridoma cell lines above may be shortened forconvenience of reference. E.g. A01.1 corresponds to Pro104.A01.1.Additionally, the names of the deposited hybridoma cell lines may or maynot contain the period punctuation mark separating “Pro104” from thehybridoma clone. E.g. Pro104.C55.1 corresponds to Pro104.C55.1. Thesehybridomas correspond to the clones (with their full names) depositedwith the ATCC. Table 10 lists the hybridoma clone deposited with theATCC, the accorded ATCC accession number, and the date of deposit. TABLE10 ATCC deposits Hybridoma ATCC Accession No. Deposit Date Pro104.C55.1PTA-5277 23 Jun. 2003 Pro104.C25.1 PTA-6076 15 June 24 2004 Pro104.D9.1PTA-6077 15 June 24 2004 Pro104.K81.15 PTA-6078 15 June 24 2004

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweendiaDexus, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws. The making of thesedeposits is by no means an admission that deposits are required toenable the invention.

1. An isolated Pro104 antibody that binds to Pro104 on a mammalian cellin vivo.
 2. The antibody of claim 1 which internalizes upon binding toPro104 on a mammalian cell in vivo.
 3. The antibody of claim 1 which isa monoclonal antibody, an antibody fragment or a chimeric or humanizedantibody. 4-5. (canceled)
 6. The antibody of claim 1 which is producedby a hybridoma selected from the group consisting of American TypeCulture Collection accession number PTA-5277, 6076, 6077 and 6078 orcompetes for binding to the same epitope as the epitope bound by amonoclonal antibody produced by a hybridoma selected from the groupconsisting of ATCC accession number PTA-5277, 6076, 6077 and
 6078. 7.(canceled)
 8. The antibody of claim 1 which is conjugated to a growthinhibitory agent or a cytotoxic agent.
 9. (canceled)
 10. The antibody ofclaim 8 wherein the cytotoxic agent is selected from the groupconsisting of toxins, antibiotics, radioactive isotopes and nucleolyticenzymes. 11-15. (canceled)
 16. An anti-Pro104 monoclonal antibody thatinhibits the growth of Pro104-expressing cancer cells in vivo. 17-18.(canceled)
 19. The antibody of claim 16, which is a humanized form of ananti-Pro104 antibody produced by a hybridoma selected from the groupconsisting of ATCC accession number PTA-5277, 6076, 6077 and
 6078. 20.The antibody of claim 16, wherein the cancer cells are from a cancerselected from the group consisting of breast, ovarian, pancreatic andlung cancer.
 21. (canceled)
 22. A cell that produces the antibody ofclaim
 1. 23. The cell of claim 22, wherein the cell is selected from thegroup consisting of hybridoma cells deposited under American TypeCulture Collection accession number PTA-5277, 6076, 6077 and 6078.24-29. (canceled)
 30. A method of killing a Pro104-expressing cancercell, comprising contacting the cancer cell with the antibody of claim1, thereby killing the cancer cell.
 31. The method of claim 30, whereinthe cancer cell is selected from the group consisting of breast,ovarian, pancreatic and lung cancer cell. 32-34. (canceled)
 35. Themethod of claim 30, wherein the antibody is an antibody fragment or ahumanized antibody.
 36. (canceled)
 37. The method of claim 30, whereinthe antibody is conjugated to a cytotoxic agent.
 38. (canceled)
 39. Themethod of claim 30, wherein the antibody is a humanized form of theantibody produced by a hybridoma selected from the group consisting ofATCC accession number PTA-5277, 6076, 6077 and
 6078. 40-46. (canceled)47. The method of claim 30, wherein the antibody is administered inconjunction with at least one chemotherapeutic agent. 48-60. (canceled)61. A method for detecting Pro104 overexpression in a subject in needthereof comprising, a) combining a serum sample of a subject with aPro104 antibody of claim 1 under conditions suitable for specificbinding of the Pro104 antibody to Pro104 in said serum sample b)determining the level of Pro104 in the serum sample, c) comparing thelevel of Pro104 determined in step b to the level of Pro104 in acontrol, wherein an increase in the level of Pro104 in the serum samplefrom the subject as compared to the control is indicative of Pro104overexpression in the subject.
 62. The method of claim 61 wherein thesubject has cancer.
 63. The method of claim 62 wherein the subject hasbreast, ovarian, pancreatic or lung cancer or a metastatic cancerthereof. 64-71. (canceled)